U.S. patent number 10,994,031 [Application Number 15/872,763] was granted by the patent office on 2021-05-04 for composites and compositions for therapeutic use and methods of making and using the same.
This patent grant is currently assigned to Board of Regents of the Nevada System of Higher Education, on behalf of the University of Nevada, Reno. The grantee listed for this patent is Board of Regents of the Nevada System of Higher Education, on behalf of the University of Nevada. Invention is credited to Violeta Demillo, Xiaoshan Zhu.
View All Diagrams
United States Patent |
10,994,031 |
Zhu , et al. |
May 4, 2021 |
Composites and compositions for therapeutic use and methods of
making and using the same
Abstract
Disclosed herein are embodiments of composites and compositions
that can be used for therapeutic applications in vivo and/or in
vitro. The disclosed composites can comprise cores having magnetic
nanoparticles, quantum dots, or combinations thereof and
zwitterionic polymeric coatings that facilitate solubility and
bioconjugation. The compositions disclosed herein can comprise the
composites and one or more biomolecules, drugs, or combinations
thereof. Also disclosed herein are methods of making the
composites, composite components, and methods of making quantum
dots for use in the composites.
Inventors: |
Zhu; Xiaoshan (Reno, NV),
Demillo; Violeta (Reno, NV) |
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents of the Nevada System of Higher Education, on
behalf of the University of Nevada |
Reno |
NV |
US |
|
|
Assignee: |
Board of Regents of the Nevada
System of Higher Education, on behalf of the University of Nevada,
Reno (Reno, NV)
|
Family
ID: |
1000005527823 |
Appl.
No.: |
15/872,763 |
Filed: |
January 16, 2018 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180154024 A1 |
Jun 7, 2018 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
PCT/US2016/042613 |
Jul 15, 2016 |
|
|
|
|
62569291 |
Oct 6, 2017 |
|
|
|
|
62203325 |
Aug 10, 2015 |
|
|
|
|
62194122 |
Jul 17, 2015 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
49/1866 (20130101); G01N 33/587 (20130101); G01N
33/588 (20130101); A61K 49/1887 (20130101); A61K
9/48 (20130101); A61K 49/0056 (20130101); A61K
49/0002 (20130101); A61K 49/0067 (20130101); B82Y
5/00 (20130101) |
Current International
Class: |
A61K
9/00 (20060101); A61K 49/00 (20060101); A61K
49/18 (20060101); A61K 9/48 (20060101); G01N
33/58 (20060101); B82Y 5/00 (20110101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO 2007/024393 |
|
Mar 2007 |
|
WO |
|
WO 2011/119654 |
|
Sep 2011 |
|
WO |
|
WO 2013/182707 |
|
Dec 2013 |
|
WO |
|
Other References
Shrake et al., "Facilitated preparation of bioconjugatable
zwitterionic quantum dots using dual-lipid encapsulation," Journal
of Colloid and Interface Science, vol. 437, pp. 140-146, Sep. 21,
2014. cited by applicant .
Zhang et al., "Self-assembly multifunctional nanocomposites with
Fe3O4 magnetic core and CdSe/ZnS quantum dots shell," Journal of
Biomedical Research Part A, pp. 840-846, Oct. 29, 2007. cited by
applicant .
Extended European Search Report issued for EPC Application No.
16828325.7 dated Jan. 24, 2019. cited by applicant .
Demillo et al., "Zwitterionic amphiphile coated magnetofluorescent
nanoparticles-synthesis, characterization and tumor cell
targeting," Journal of Materials Chemistry B, 3(42): 8328-8336,
Sep. 14, 2015. cited by applicant .
Examination Report issued by European Patent Office dated Nov. 6,
2019, for EPC Application No. 16828325.7. cited by applicant .
International Search Report and Written Opinion issued for
International Application No. PCT/US2016/042613 dated Oct. 18,
2016. cited by applicant .
Chen et al., "Thermal decomposition based synthesis of
Ag--In--S/Zns quantum dots and their chlorotoxin-modified micelles
for brain tumor cell targeting," RSC Adv., 74(5): 60612-60620, Jul.
8, 2015. cited by applicant .
Huang et al., "A polymer encapsulation approach to prepare
zwitterion-like, biocompatible quantum dots with wide pH and ionic
stability," J. Nanopart. Res., 16(8): 2555, Jul. 19, 2014. cited by
applicant .
Demillo et al., "Fabrication of MnFe.sub.2O.sub.4--CuInS.sub.2/ZnS
magnetofluorescent nanocomposites and their characterization,"
Colloids and Surfaces A: Physicochemical and Engineering Aspects,
464(5): 134-142, Oct. 16, 2014. cited by applicant .
Cormode et al., "A versatile and tunable coating strategy allows
control of nanocrystal delivery to cell types in the liver,"
Bioconjug. Chem., 22(3): 353-361, Mar. 16, 2011. cited by applicant
.
Tarannum et al., "Advances in synthesis and applications of sulfo
and carbo analogues of polybetaines: a review," Reviews in Advanced
Sciences and Engineering, 2(2): 90-111, Jun. 2013. cited by
applicant .
Han et al., "Spatial charge configuration regulates nanoparticle
transport and binding behavior in vivo," Angew Chem Int Ed Engl.,
52(5): 1414-1419, Jan. 28, 2013. cited by applicant .
Wu et al., "Carboxybetaine, sulfobetaine, and cationic block
copolymer coatings: a comparison of the surface properties and
antibiofouling behavior," Journal of Applied Polymer Science,
124(3): 2154-2170, May 5, 2012. cited by applicant .
Kober et al., "Transient magnetic birefringence for determining
magnetic nanoparticle diameters in dense, highly light scattering
media," Nanotechnology, 23(15): 25 pages, Mar. 28, 2012. cited by
applicant .
Jiang et al., "An effective polymer cross-linking strategy to
obtain stable dispersions of upconverting NaYF.sub.4 nanoparticles
in buffers and biological growth media for biolabeling
applications," Langmuir, 28(6): 3329-3247, Jan. 17, 2012. cited by
applicant.
|
Primary Examiner: Dickinson; Paul W
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Government Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with government support under Contract No.
1P20GM103650 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application
No. 62/569,291, filed on Oct. 6, 2017; and is a
continuation-in-part of PCT Application No. PCT/US2016/042613,
filed on Jul. 15, 2016, which claims the benefit of U.S.
Provisional Application No. 62/203,325, filed Aug. 10, 2015, and
U.S. Provisional Application No. 62/194,122, filed on Jul. 17,
2015; each of these applications is incorporated herein by
reference in its entirety.
Claims
We claim:
1. A composite, comprising: a core comprising one or more magnetic
nanoparticles, one or more quantum dots, or a combination thereof;
and a zwitterionic polymeric coating defining the core and
comprising a zwitterionic polymer having a structure satisfying a
formula ##STR00022## wherein each R.sup.1 independently is hydrogen
or aliphatic; each R.sup.2 independently is --C(O)Z, wherein Z is
hydroxyl, ether, amine, thiol, or thioether; at least one R.sup.3
is amide-aliphatic-amine-aliphatic-carboxylate or
amide-aliphatic-amine-aliphatic-sulfonate wherein the amine is
positively charged, and each other R.sup.3 independently is
amide-aliphatic-amine, amide-aliphatic-amine-aliphatic-carboxylate,
amide-aliphatic-amine-aliphatic-sulfonate, or
amide-aliphatic-thiol; each p independently is an integer selected
from zero to 5; q is an integer selected from zero or 1; and each
of a and b independently is an integer selected from 1 to 200.
2. The composite of claim 1, wherein the zwitterionic polymeric
coating comprises a zwitterionic polymer having a structure
satisfying a formula selected from ##STR00023## wherein each
R.sup.1 independently is hydrogen or aliphatic; each of X.sup.1,
X.sup.2, X.sup.3, X.sup.4, and X.sup.5 independently is NR.sup.b,
N(R.sup.b).sub.2+, oxygen, or sulfur, wherein each R.sup.b
independently is hydrogen, aliphatic, heteroaliphatic, aryl, or
heteroaryl; each of A.sup.1, A.sup.2, A.sup.3, A.sup.4, and A.sup.5
independently is aliphatic or heteroaliphatic; each of Y.sup.1,
Y.sup.2, and Y.sup.3 independently is amine, thiol, carboxylate or
sulfonate; each Z independently is hydroxyl, ether, amine, thiol,
or thioether; n is an integer selected from 1 to 200; each m
independently is an integer selected from 0 to 3; each p
independently is an integer selected from zero to 5; each of a, b,
and c independently is an integer selected from 1 to 200.
3. The composite of claim 2, wherein each R.sup.1 independently is
hydrogen, alkyl, alkenyl, or alkynyl; each of X.sup.1, X.sup.2,
X.sup.3, X.sup.4, and X.sup.5 independently is NR.sup.b,
N(R.sup.b).sub.2+, oxygen, or sulfur, wherein each Rb independently
is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, phenyl, naphthyl, or pyridinyl; each of A.sup.1,
A.sup.2, A.sup.3, A.sup.4, and A.sup.5 independently is alkyl,
alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl;
each of Y.sup.1 and Y.sup.2 independently is carboxylate or
sulfonate and Y.sup.3 is thiol; each Z is hydroxyl; and m is 1.
4. The composite of claim 1, wherein the zwitterionic polymeric
coating comprises a zwitterionic polymer have a structure selected
from ##STR00024## ##STR00025## wherein d is an integer selected
from 1 to 200.
5. The composite of claim 1, wherein the core comprises at least
one magnetic nanoparticle and at least one quantum dot.
6. The composite of claim 1, wherein the core is doped with a metal
selected from Cu, Ag, Au, Mn, Zn, or combinations thereof and
wherein the core is doped with greater than 0 mol % to 10 mol % of
the metal.
7. The composite of claim 6, wherein the core is a I-II-VI quantum
dot core and the quantum dot core comprises a ZnS or a chloride
shell.
8. The composite of claim 1, wherein the magnetic nanoparticle is
MnFe.sub.2O.sub.4, Fe.sub.3O.sub.4, CoFe.sub.2O.sub.4, FePt or a
combination thereof.
9. The composite of claim 1, wherein the core comprises a
combination of MnFe.sub.2O.sub.4 nanoparticles and an AgInS.sub.2
quantum dot, a CuInS.sub.2 quantum dot, or a combination
thereof.
10. The composite of claim 1, comprising: a core comprising a
combination of MnFe.sub.2O.sub.4 nanoparticles and CuInS.sub.2
quantum dots, AgInS.sub.2 quantum dots, or both CuInS.sub.2 quantum
dots, AgInS.sub.2 quantum dots; and a zwitterionic polymeric
coating comprising a zwitterionic polymer having a structure
selected from ##STR00026## ##STR00027## wherein d is an integer
ranging from 1 to 200.
11. The composite of claim 10, wherein the core further comprises a
dopant selected from Cu, Ag, Au, Mn, Zn, or combinations thereof, a
ZnS or chloride shell, or both the dopant and the ZnS or chloride
shell.
12. A composition comprising: a composite having a core comprising
one or more magnetic nanoparticles, one or more quantum dots, or a
combination thereof and a zwitterionic polymeric coating comprising
a zwitterionic polymer having a structure satisfying a formula
##STR00028## wherein each R.sup.1 independently is selected from
hydrogen or aliphatic; each R.sup.2 independently is --C(O)Z,
wherein Z is selected from hydroxyl, ether, amine, thiol, or
thioether; at least one R.sup.3 is
amide-aliphatic-amine-aliphatic-carboxylate or
amide-aliphatic-amine-aliphatic-sulfonate wherein the amine is
positively charged, and each other R.sup.3 independently is
selected from amide-aliphatic-amine,
amide-aliphatic-amine-aliphatic-carboxylate,
amide-aliphatic-amine-aliphatic-sulfonate, or
amide-aliphatic-thiol; each p independently is an integer selected
from zero to 5; q is an integer selected from zero or 1; and each
of a and b independently is an integer selected from 1 to 200; and
a biomolecule, a drug, or a combination thereof.
13. The composition of claim 12, wherein the biomolecule is
chemically conjugated to the composite through the zwitterionic
polymeric coating or through a carboxylate group of the
zwitterionic polymeric coating.
14. The composition of claim 13, wherein the carboxylate group of
the zwitterionic polymeric coating is further chemically bound to a
linker, wherein the linker is also chemically bound to the
biomolecule.
15. The composition of claim 12, wherein the biomolecule is
selected from chlorotoxin, avidin, biotin, folic acid,
arginylglycylaspartic acid, or combinations thereof.
16. The composition of claim 12, wherein the drug is encapsulated
within the zwitterionic polymeric coating and is selected from
doxorubicin, daunorubicin, epirubicin, idarubicin, or a combination
thereof.
17. A method of making the composite of claim 1, comprising:
combining, to form a mixture, a solution comprising the one or more
magnetic nanoparticles, the one or more quantum dots, or the
combination thereof with a solution of the zwitterionic polymer;
dispersing the mixture into water using sonication to form a
dispersed composition; and isolating the composite from the
dispersed composition.
18. A method for imaging cells, comprising: contacting a cell with
the composite of claim 1; and detecting cellular update and/or the
location of the composite in the cell.
19. A method of delivering a therapeutic drug to a subject,
comprising contacting the subject with a therapeutically effective
amount of a composition comprising the therapeutic drug and the
composite of claim 1.
Description
FIELD
The present disclosure concerns embodiments of composites and
compositions comprising nanoparticle and/or quantum dot cores, and
zwitterionic polymeric coatings. Also disclosed herein are
embodiments of methods of making components used in the composites
and compositions, as well as methods of making and using the
composites and compositions.
BACKGROUND
Magnetofluorescent nanoparticles enabling simultaneous fluorescence
labeling and magnetic field assisted separation, sorting, heating
or imaging are gaining momentum for biomedical applications at the
cellular, tissue or anatomical levels. For this reason, research
approaches have been reported on the controlled synthesis of
magnetofluorescent nanoparticles regarding their size, shape,
composite and surface properties. To achieve colloidal stability
and biocompatibility for various in vitro or in vivo applications,
the hydrophilic shell of these type of magnetofluorescent
nanoparticles are usually formed by anti-fouling poly(ethylene
glycol) (PEG) chains. Zwitterion-coating approaches also have been
developed for individual cadmium-based quantum dots or AuNPs but
not for magnetofluorescent compounds. These approaches mainly
involve the coupling of zwitterions with thiol ligands, such as
dihydrolipoic acid. These coupled ligands are further exchanged
with native hydrophobic ligands (trioctylphosphine oxide) to form
thiol-bound zwitterionic quantum dots through the high binding
affinity of thiols to Zn or Au atoms on quantum dot or AuNP
surface.
Significant efforts are still needed to develop high quality
quantum dots into broader biomedical applications especially for in
vitro or in vivo sensing/imaging and drug delivery. There exists a
need in the art for facile, scalable methods of making chalcopyrite
quantum dots that are compatible for use in magnetofluorescent
composites comprising nanoparticles. There also exists a need in
the art for composites comprising zwitterionic coatings that can be
used to produce composites capable for biomedical use and/or
coupling to biomolecules.
SUMMARY
Disclosed herein are embodiments of composites comprising a core
comprising one or more nanoparticles (such as magnetic
nanoparticles), one or more quantum dots, or a combination thereof,
and a zwitterionic polymeric coating comprising a zwitterionic
polymer having any one of the formulas and/or structures disclosed
herein. In some embodiments, the core comprises at least one
magnetic nanoparticle and at least one quantum dot. Also disclosed
herein are embodiments of compositions comprising a composite
having a core comprising one or more magnetic nanoparticles, one or
more quantum dots, or a combination thereof, a zwitterionic
polymeric coating defining the core, and a biomolecule, a drug, or
a combination thereof.
Also disclosed herein are embodiments of methods for making the
composites, methods of making quantum dots, and methods of using
the disclosed composites for visualizing uptake/binding in cells or
tissues and/or treating subjects.
The foregoing and other objects, features, and advantages of the
present disclosure will become more apparent from the following
detailed description, which proceeds with reference to the
accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a combined photoluminescence spectrum of AgInS.sub.2
(AIS) quantum dots and AIS/ZnS quantum dots where the inset shows a
digital photograph of AIS quantum dots and AIS/ZnS quantum dots in
organic solvents exposed under a UV laser beam.
FIG. 2 is a combined Fourier Transform Infrared (FT-IR) spectrum of
a representative polymer precursor ("PMAO"), a representative
polymer intermediate ("PMAO-DMAPA"), and a zwitterionic polymer
("PMAO-CB-SB").
FIG. 3 is a combined FT-IR spectra of amidosulfobetaine-16
("ASB-16") (with sulfobetaine) and
N-dodecyl-N,N-(dimethylammonio)butyrate ("DDMAB") (with
carboxybetaine).
FIG. 4 is a graph of normalized photoluminescence intensity (a.u.)
as a function of wavelength (nm) illustrating representative and
normalized photoluminescence intensity of CIS quantum dots in the
time course of growth at 240.degree. C.
FIG. 5 is a combined .sup.1H-NMR spectrum illustrating NMR spectra
of a representative polymer precursor ("PMAO"), a representative
polymer intermediate ("PMAO-DMAPA"), and a zwitterionic polymer
("PMAO-CB-SB").
FIGS. 6A-6C illustrate TEM and EDX images of representative
composite cores; FIG. 6A is a TEM image of a representative
composite core having a nanoparticle to quantum dot ratio of 1:4;
FIG. 6B is a zoomed image of a single composite core illustrating
both spherical and irregular particle shapes representing
nanoparticles and quantum dots; and FIG. 6C is an EDX spectrum.
FIGS. 7A and 7B are graphs illustrating results obtained from
analysis of representative composites; FIG. 7A is a combined
photoluminescence spectrum of hydrophobic quantum dots in organic
solvent and water-soluble composites as disclosed herein; FIG. 7B
is a graph of R.sub.2 parameters of composites disclosed herein
versus (Mn+Fe) concentration (the slope r.sub.2 of each
curve=R.sub.2/[(Mn+Fe) concentration]), wherein the inset image is
a representative magnetic resonance image of zwitterionic
magnetofluorescent nanoparticles and zwitterionic magnetic
nanoparticles.
FIGS. 8A and 8B illustrate results obtained from analysis of
representative composites; FIG. 8A is a combined spectrum
illustrating photoluminescence of hydrophobic quantum dots in THF
and water-soluble composites; and FIG. 8B is a graph of R.sub.2
parameters of composites versus (Mn+Fe) concentration (the slope
r.sub.2 of each curve=R.sub.2/[(Mn+Fe) concentration]), wherein the
inset image is a representative magnetic resonance image of
composites fabricated with the nanoparticle to quantum dot mass
ratios of 1:0, 1:1, 1:2, and 1:4.
FIG. 9 illustrates the UV-Vis absorbance spectra of quantum dots in
THF and representative water-soluble composites.
FIG. 10 is a bar graph of fluorescence stability of representative
composites in PBS solutions at pH values ranging from 4-9, which
illustrates the colloidal stability of the composites in aqueous
solutions.
FIG. 11 is a bar graph of fluorescence stability of representative
composites in water at 4.degree. C.
FIG. 12 is a bar graph of long-term stability of representative
composites in water at 4.degree. C.
FIGS. 13A and 13B are graphs illustrating cell viability cell
treated with representative composites disclosed herein
illustrating that the composites have no significant effects on
cell cytotoxicity; FIG. 13A shows cell viability of U-87 MG cells
treated with composites at different concentrations over 24 hours;
and FIG. 13B shows cell viability of HEK-293 cells treated with
zwitterionic magnetofluorescent nanoparticles at different
concentrations over 24 hours.
FIG. 14 is a graph illustrating cell viability of U-87 MG cell and
HEK-293 cells treated with representative composites at difference
concentrations over 24 hours.
FIG. 15 is a bar graph illustrating the fluorescence responses to
biotinylated magnetic microbeads after incubation with serial
dilutions of non-conjugated and Neutravidin-conjugated composites
from their stock suspensions.
FIGS. 16A-16E illustrate results obtained for representative
composites disclosed herein; FIG. 16A is a confocal image
illustrating cellular uptake/internalization of cyclophosphamide
("CTX")-conjugated composites by U-87 cells; FIG. 16B is a confocal
image illustrating cellular uptake/internalization of
non-conjugated composites by U-87 cells; FIG. 16C is a confocal
image illustrating cellular uptake/internalization of
CTX-conjugated composites by HEK-293; FIG. 16D is a confocal image
illustrating cellular uptake/internalization of non-conjugated
composites by HEK-293 cells; and FIG. 16E is a bar graph of micelle
photoluminescence intensity as a function of composite
concentration illustrating the fluorescent intensity per unit area
of cytoplasm for U-87 and HEK-293 cells incubated with CTX- and
non-conjugated composites with different concentrations.
FIGS. 17A-17D are confocal images at different channels and their
overlays demonstrating the cellular uptake/internalization of
CTX-conjugated composites by U-87 (FIG. 17A) and HEK-293 (FIG. 17C)
and non-conjugated composites under the same concentration of
particles by U-87 (FIG. 17B) and HEK-293 (FIG. 17D).
FIG. 18 illustrates the evolution of the photoluminescence spectra
of a representative quantum dot used in composites described herein
during the time course of growth at 170.degree. C.
FIGS. 19A-19C are images illustrating results obtained from TEM and
EDX analysis of representative quantum dots made using methods
described herein; FIG. 19A is a TEM image of a representative
quantum dot; FIG. 19B is a high resolution TEM image of a
representative quantum dot; and FIG. 19C is an EDX spectrum of a
representative quantum dot.
FIG. 20 is an EDX spectrum of a representative quantum dot made
using a thermal decomposition synthesis embodiment.
FIG. 21 illustrates XRD patterns for representative shell-coated
and shell-free quantum dots; diffraction peaks of tetragonal
AgInS.sub.2 and cubic ZnS are shown as references.
FIG. 22 illustrates photoluminescence spectra of representative
quantum dots made using a disclosed thermal decomposition method
embodiment.
FIG. 23 illustrates UV-Vis absorption spectra representative
quantum dots synthesized at different ratios (1:1, 1:2 and 1:4),
wherein the inset provides digital photograph of the quantum dots
under room lights.
FIGS. 24A and 24B are digital photographs of representative quantum
dots made using a disclosed thermal decomposition method
embodiment; FIG. 24A illustrates quantum dots made using a Ag:In
molar ratio of Ag:In=1:1; and FIG. 24B illustrates quantum dots
made using a Ag:In molar ratio of Ag:In=1:2.
FIGS. 25A and 25B are graphs illustrating the photostability of AIS
quantum dots (FIG. 25A) and AIS/ZnS quantum dots (FIG. 25B),
wherein quantum dots in organic solvents were under continuous
exposure of a 365 nm UV lamp for 120 minutes and their
photoluminescence was measured every 30 minutes.
FIG. 26 is a schematic illustration of quantum dot-loaded micelles
comprising quantum dots made using thermal decomposition method
embodiments disclosed herein.
FIG. 27 illustrates photoluminescence spectra of representative
quantum dots in hexane and quantum dot micelles in water, wherein
the insets are the hydrodynamic size distribution of the resulting
quantum dot-micelles measured by DSL (top) and the corresponding
TEM image of the quantum dot-micelles.
FIGS. 28A-28C are TEM and EDX images obtained for representative
quantum dots; FIG. 28A is a TEM image of an individual quantum
dot-micelle; FIG. 28B is a high resolution TEM image of an
individual quantum dot-micelle; and FIG. 28C is an EDX spectrum of
quantum dot-micelles.
FIG. 29 illustrates FT-IR spectra of representative quantum dots,
PLGA-PEG, CTX, quantum dot-micelles, and CTX-conjugated quantum
dot-micelles.
FIGS. 30A-30C illustrate results obtained from analysis of
representative quantum dots made using a disclosed thermal
decomposition method embodiment; FIG. 30A shows confocal images
demonstrating the cellular uptake/internalization of CTX-conjugated
quantum dot-micelles (100 times diluted from stock) by U-87 cells;
FIG. 30B shows confocal images presenting the quenching effect of
1,10-phenanthroline on the uptake/internalization; and FIG. 30C
shows quantitative data indicating the cellular
uptake/internalization and the quenching effect under the different
concentrations or dilutions of CTX-conjugated quantum dot-micelles
(100-800 times dilutions).
FIGS. 31A-31E illustrate results obtained from analysis of
representative quantum dots made using a disclosed thermal
decomposition methods embodiment; FIG. 31A is an overlaid confocal
image of U-87 cells after incubation with CTX-conjugated micelles
comprising the quantum dots; FIG. 31B is an overlaid confocal image
of U-87 cells after incubation with non-conjugated micelles
comprising the quantum dots; FIG. 31C is an overlaid confocal image
of HEK-293 cells after incubating with CTX-conjugated micelles
comprising the quantum dots; FIG. 31D is an overlaid confocal image
of HEK-293 cells after incubating with non-conjugated micelles
comprising the quantum dots; and FIG. 31E illustrates the
fluorescent intensity per unit area of cytoplasm for U-87 and
HEK-293 cells incubated with CTX-conjugated and non-conjugated
AIS/ZnS micelles.
FIG. 32 provides an exemplary schematic of a representative method
of making micelles comprising the polymeric coatings disclosed
herein.
FIG. 33 is a schematic diagram illustrating a representative
composite and use of the composite to administer drug molecules to
a cell.
FIGS. 34A and 34B each are TEM images of a representative quantum
dot made using a disclosed thermal decomposition method
embodiment.
FIGS. 35A and 35B show results obtained from analyzing
representative doped quantum dots; FIG. 35A is a graph of
photoluminescence intensity (a.u.) as a function of wavelength (nm)
showing the effect of Cu-dopant concentrations on photoluminescence
for surface-doped quantum dots, wherein the inset data plot shows
that the evolution of photoluminescence spectra of non-doped AIS
nanocrystals is not significant in the time course of growth; and
FIG. 35B shows the absorption spectra of the copper-doped quantum
dots with different Cu initial concentrations.
FIGS. 36A and 36B show results obtained from time course analysis
of Cu-doped quantum dots; FIG. 36A shows the evolution of
photoluminescence spectra of 3.33% Cu:AIS quantum dots in the time
course of reaction using a surface doping approach, wherein the
inset is the evolution of photoluminescence spectra of Cu:AIS
quantum dots prepared through a homogenous reaction; FIG. 36B shows
the temporal evolution of absorption spectra of 3.33% Cu:AIS
quantum dots.
FIGS. 37A-37D show characterization data of representative Cu-doped
quantum dots disclosed herein; FIG. 37A shows XRD patterns for AIS,
1.67% Cu:AIS and 6.67% Cu:AIS quantum dots; FIG. 37B shows TEM and
high resolution TEM (insets) images of AIS quantum dots; FIG. 37C
shows TEM and high resolution TEM (insets) images of 1.67% Cu:AIS
quantum dots; and FIG. 37D shows TEM and high resolution TEM
(insets) images of 6.67% Cu:AIS quantum dots.
FIGS. 38A-38D show results obtained from characterization analysis
of representative embodiments of Cu-doped core quantum dots and
Cu-doped core/shell quantum dots; FIG. 38A shows digital images of
Cu:AIS and Cu:AIS/ZnS quantum dots suspended in organic solvents
under a UV lamp;
FIG. 38B is a combined absorption spectrum of photoluminescence and
absorption spectra of the core and core/shell quantum dots; FIG.
38C is shows the XRD patterns of 6.67% Cu:AIS and Cu:AIS/ZnS
quantum dots; FIG. 38D shows TEM and high resolution TEM (inset)
images of 6.67% Cu:AIS/ZnS quantum dots.
FIGS. 39A and 39B are photoluminescence decay curves showing the
photoluminescence decay for representative Cu:AIS quantum dots
(FIG. 39A) and Cu:AIS/ZnS quantum dots (FIG. 39B).
FIG. 40 is a graph of cell viability (%) as a function of composite
(referred to as "micelle") concentration showing cell viability of
U-87 MG cell and HEK-293 cells treated with representative
composite embodiments at different concentrations over 48
hours.
FIGS. 41A-41F are overlaid confocal images demonstrating the
cellular uptake/internalization of RGD-conjugated, RAD-conjugated,
and non-conjugated composites under the same dilution or
concentration of composites (25 times dilution from the conjugate
stock) by U-87 (FIGS. 41A-41C) and HEK-293 (FIGS. 41D-41F).
FIG. 42 is a graph of composite (or "micelle") photoluminescence
intensity as a function of composite ("micelle") concentration
showing the fluorescent intensity per unit area of cytoplasm for
U-87 and HEK-293 cells incubated with non-conjugated micelles,
RAD-conjugated micelles, and RGD-conjugated composites with
different dilutions or concentrations (all p values for each
comparison are less than 0.001).
FIG. 43 is an illustration of a sensing probe as described
herein.
FIGS. 44A and 44B are spectra showing absorbance and
photoluminescence intensity for composites comprising chloride
shells (FIG. 44A) and control examples without chloride shells
(FIG. 44B).
FIGS. 45A-45C illustrate results obtained from analyzing Mn-doped
nanocrystals as described herein, wherein the surface doping is
used; FIG. 45A shows a representative synthesis of Mn:AIZS-ZnS
nanocrystal embodiments and the effect of Mn levels on
photoluminescence tunability, wherein AIZS cores are formed first,
Mn and Zn precursors are added to the surface, and the nanocrystal
surfaces are further passivated by zinc (yellow, red, and green
represent AIZS composites, Mn, and ZnS shell, respectively); FIG.
45B is a graph showing photoluminescence spectra of Mn:AIZS/ZnS
nanocrystals as compared to an undoped AIZS/ZnS nanocrystal; and
FIG. 45C is a graph showing absorption spectra of the nanocrystals,
wherein some there is some spectrum overlap between
photoluminescence spectra and absorption spectra indicating some
photoluminescence emissions will be reabsorbed by the nanocrystals
themselves.
FIG. 46 illustrates XRD patterns of an undoped AIS/ZnS nanocrystal,
a 0.025 mmol-Mn-doped AIS/ZnS nanocrystal, a 0.075 mmol-Mn-doped
AIS/ZnS nanocrystal, and a 0.125 mmol-Mn-doped AIS/ZnS nanocrystal,
wherein diffraction peaks of tetragonal AgInS.sub.2 and ZnS, as
obtained from the JCPDS database (specifically, JCPDS #25-1330 and
JCPDS #05-0566), are shown as references.
FIGS. 47A-47D illustrate images of a Mn-doped nanocrystal
embodiments, wherein FIG. 47A is a TEM image of a 0.075
mmol-Mn-doped AIZS/ZnS nanocrystal; FIG. 47B is a high resolution
TEM image of a 0.075 mmol-Mn-doped AIZS/ZnS nanocrystal; FIG. 47C
is a fast Fourier transform (FFT) pattern on the HRTEM image of a
0.075 mmol-Mn-doped AIZS/ZnS nanocrystal; and FIG. 47D is an EDX
spectrum of the 0.075 mmol-Mn-doped AIZS/ZnS nanocrystal, which
illustrates Ag, In, Mn, Zn and S elements (and wherein the
unlabeled peaks above 10 KeV are gold elements from the gold-mesh
grid used for TEM imaging).
FIG. 48 is a graph illustrating photoluminescence decay curves of a
0.075 mmol-Mn-doped AIZS/ZnS nanocrystal, wherein the sample is
excited at 300 nm by a xenon lamp and its photoluminescence decays
were measured at different wavelengths.
FIG. 49 is a collection of photographic images illustrating results
obtained from a thermal stability study of Mn:AIZS/ZnS nanocrystal,
wherein the Mn:AIZS nanocrystal solutions are shown at room
temperature, after heating at 170.degree. C., and cooled down to
room temperature under a UV lamp.
FIGS. 50A-50C are images illustrating results from analyzing a
Mn-doped nanocrystal embodiment wherein the core of the nanocrystal
is doped; FIG. 50A is an illustration of such a nanocrystal wherein
Mn atoms are doped into the core of an AZIS nanocrystal and then
the cores are shelled with ZnS layers; FIG. 50B is a graph showing
fluorescence and absorption spectra of Mn-doped AZIS/ZnS
nanocrystals; and FIG. 50C is a graph showing fluorescence decays
of Mn-doped AZIS/ZnS nanocrystals under different Ag levels in the
composites.
FIGS. 51A and 51B provide a schematic illustration of a
representative composite wherein a zwitterionic polymer
encapsulates both magnetic nanoparticles and I(II)-III-VI quantum
dots through self-assembly (FIG. 51B) and representative magnetic
resonance image and optical image of the composite at difference
concentrations, wherein the concentration is marked by the Fe+Mn
mass/mL (FIG. 51B).
FIG. 52 is a graph illustrating results obtained from cell
viability testing, wherein unconjugated and chlorotoxin-conjugated
conjugates prepared using composite embodiments described herein
were incubated with cells over 72 hours.
FIGS. 53A and 53B are images showing intracellular distribution of
conjugated micelles in U-87, wherein micelles, endosomes or
lysosomes are stained with Lysotracker green, and nuclei are
stained with DAPI appear red, green, and blue, respectively; FIG.
53A shows distribution after 3 hours of incubation; and FIG. 53B
shows distribution after 24 hours of incubation.
FIGS. 54A-54C shows results obtained from measuring the
fluorescence lifetime of a control (organic dye C153) and two
composite embodiments described herein, namely an Mn-doped AZIS/ZnS
composite having an average lifetime of 0.1 ms (FIG. 54B) and
another embodiment having an average lifetime of 1 ms (FIG. 54C),
wherein the insets show the zero backgrounds.
DETAILED DESCRIPTION
I. Explanation of Terms
The following explanations of terms are provided to better describe
the present disclosure and to guide those of ordinary skill in the
art in the practice of the present disclosure. As used herein,
"comprising" means "including" and the singular forms "a" or "an"
or "the" include plural references unless the context clearly
dictates otherwise. The term "or" refers to a single element of
stated alternative elements or a combination of two or more
elements, unless the context clearly indicates otherwise.
Unless explained otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present disclosure, suitable methods and materials are described
below. The materials, methods, and examples are illustrative only
and not intended to be limiting, unless otherwise indicated. Other
features of the disclosure are apparent from the following detailed
description and the claims.
Unless otherwise indicated, all numbers expressing quantities of
components, molecular weights, percentages, temperatures, times,
and so forth, as used in the specification or claims are to be
understood as being modified by the term "about." Accordingly,
unless otherwise indicated, implicitly or explicitly, the numerical
parameters set forth are approximations that can depend on the
desired properties sought and/or limits of detection under standard
test conditions/methods. When directly and explicitly
distinguishing embodiments from discussed prior art, the embodiment
numbers are not approximates unless the word "about" is recited.
Furthermore, not all alternatives recited herein are
equivalents.
To facilitate review of the various embodiments of the disclosure,
the following explanations of specific terms are provided. Certain
functional group terms include an R.sup.a group that, though not
part of the defined functional group, indicates how the functional
group attaches to the compound to which it is bound.
Aliphatic: A hydrocarbon, or a radical thereof, having at least one
carbon atom to 50 carbon atoms, such as one to 25 carbon atoms, or
one to ten carbon atoms, and which includes alkanes (or alkyl),
alkenes (or alkenyl), alkynes (or alkynyl), including cyclic
versions thereof, and further including straight- and
branched-chain arrangements, and all stereo and position isomers as
well.
Alkyl: A saturated monovalent hydrocarbon having at least one
carbon atom to 50 carbon atoms, such as one to 25 carbon atoms, or
one to ten carbon atoms, wherein the saturated monovalent
hydrocarbon can be derived from removing one hydrogen atom from one
carbon atom of a parent compound (e.g., alkane). An alkyl group can
be branched, straight-chain, or cyclic (e.g., cycloalkyl).
Alkenyl: An unsaturated monovalent hydrocarbon having at least two
carbon atoms to 50 carbon atoms, such as two to 25 carbon atoms, or
two to ten carbon atoms, and at least one carbon-carbon double
bond, wherein the unsaturated monovalent hydrocarbon can be derived
from removing one hydrogen atom from one carbon atom of a parent
alkene. An alkenyl group can be branched, straight-chain, cyclic
(e.g., cycloalkenyl), cis, or trans (e.g., E or Z).
Alkynyl: An unsaturated monovalent hydrocarbon having at least two
carbon atoms to 50 carbon atoms, such as two to 25 carbon atoms, or
two to ten carbon atoms and at least one carbon-carbon triple bond,
wherein the unsaturated monovalent hydrocarbon can be derived from
removing one hydrogen atom from one carbon atom of a parent alkyne.
An alkynyl group can be branched, straight-chain, or cyclic (e.g.,
cycloalkynyl).
Aryl: An aromatic carbocyclic group comprising at least five carbon
atoms to 15 carbon atoms, such as five to ten carbon atoms, having
a single ring or multiple condensed rings, which condensed rings
can or may not be aromatic provided that the point of attachment is
through an atom of the aromatic carbocyclic group.
Carboxylate: R.sup.aC(O)O.sup.-, wherein R.sup.a is the atom of the
formulas disclosed herein to which the carboxyl group is
attached.
Composite: A material comprising a core (which may comprise a
nanoparticle, a quantum dot having a core or core/shell structure,
or a combination or plurality thereof) and a zwitterionic polymeric
coating comprising a zwitterionic polymer having a structure
satisfying a formula disclosed herein, wherein the zwitterionic
polymer coating encapsulates the core and wherein the composite
exhibits properties that differ from each of its individual
components.
Encapsulated: When a drug is described herein as "encapsulated," it
is intended to mean that the drug is located within or attached to
polymer side chains of the zwitterionic polymeric coatings
described herein and/or within the core of the composite as defined
by the zwitterionic polymeric coating.
Heteroaliphatic: An aliphatic group comprising at least one
heteroatom to 20 heteroatoms, such as one to 15 heteroatoms, or one
to 5 heteroatoms, which can be selected from, but not limited to
oxygen, nitrogen, sulfur, selenium, phosphorous, and oxidized forms
thereof within the group.
Heteroalkyl/Heteroalkenyl/Heteroalkynyl: An alkyl, alkenyl, or
alkynyl group (which can be branched, straight-chain, or cyclic)
comprising at least one heteroatom to 20 heteroatoms, such as one
to 15 heteroatoms, or one to 5 heteroatoms, which can be selected
from, but not limited to oxygen, nitrogen, sulfur, selenium,
phosphorous, and oxidized forms thereof within the group.
Heteroaryl: An aryl group comprising at least one heteroatom to six
heteroatoms, such as one to four heteroatoms, which can be selected
from, but not limited to oxygen, nitrogen, sulfur, selenium,
phosphorous, and oxidized forms thereof within the ring. Such
heteroaryl groups can have a single ring or multiple condensed
rings, wherein the condensed rings may or may not be aromatic
and/or contain a heteroatom, provided that the point of attachment
is through an atom of the aromatic heteroaryl group.
Magnetic Nanoparticle: A class of nanoparticles that can be
manipulated using magnetic field gradients.
Quantum Dot: A nanoscale particle that exhibits size-dependent
electronic and optical properties due to quantum confinement. The
quantum dots disclosed herein generally have at least one dimension
less than 100 nanometers. The disclosed quantum dots may be
colloidal quantum dots, i.e., quantum dots that may remain in
suspension when dispersed in a liquid medium.
Sulfonate: A functional group having a formula
R.sup.aSO.sub.3.sup.-, wherein R.sup.a is the atom of the formulas
disclosed herein to which the sulfonate is attached.
Zwitterionic Polymer/Zwitterionic Polymeric Coating: A polymer or
polymeric coating disclosed herein that comprises both positive and
negative electrical charges and that comprises a polymeric backbone
having one or more zwitterionic side-chains extending therefrom.
Typically, the polymers and polymeric coatings comprise at least
one functional group that comprises a positive charge and at least
one functional group that comprises a negative charge, and in most
embodiments both such functional groups are located in the same
polymer side chain (as distinguished from the polymer
backbone).
II. Introduction
Micellar magnetofluorescent nanoparticles inherit the merits of
magnetic nanoparticles (e.g., high saturation magnetization) and
quantum dots (e.g., photostability and luminescence wavelength
tunability), and also provide complementary merits from both
magnetic resonance imaging and optical imaging (i.e., high spatial
resolution and high sensitivity). Conventional methods used to
produce magnetofluorescent nanoparticles require forming a
hydrophilic shell using anti-fouling poly(ethylene glycol) (PEG)
chains to achieve colloidal stability and biocompatibility for
various in vitro or in vivo applications. Although PEG chains
render such nanoparticles stable in physiological media, PEG groups
are sensitive to solution pH and salinity and tend to cause such
nanoparticles to aggregate in acidic or salt-rich
microenvironments. This aggregation further degrades and can even
change the diagnosis/therapy functionalities devised for original
nanoparticles. This shortcoming of PEG-functionalized
magnetofluorescent nanoparticles limits their applications in
biological or biomedical applications, where harsh conditions are
ubiquitous. For instance, many cellular organelles are maintained
under acidic conditions and rich in salts. Moreover, it has been
suggested in the art that PEG may induce in vivo production of
anti-PEG immunoglobulin M (IgM) antibodies, which further affects
in vivo applications of PEG coated magnetofluorescent
nanoparticles.
Other conventional magnetofluorescent nanoparticles comprise
cadmium-based quantum dots and/or gold nanoparticles with
dihydrolipoic acid that can undergo ligand exchange with
trioctylphosphine oxide ligands to form zwitterionic cadmium-based
quantum dots or gold nanoparticles with singular ligands that are
coupled to the cadmium core through a sulfur atom. Zwitterion
coupled thiol ligands, however, have not been applicable to the
preparation of magnetofluorescent nanoparticles integrating both
magnetic nanoparticles and quantum dots. These thiol ligands are
individually attached to the core and do not comprise a polymeric
backbone.
The disclosed composites and methods of making the same address
deficiencies associated with conventional composites and
techniques. For example, scalable syntheses of high quality quantum
dots are desired because a large amount of bright quantum dots can
be produced in a single synthetic reaction to sustain biomedical
research for reliable experimental observation or data collection
and also save synthesis costs. The disclosed methods provide the
ability to obtain such scalable syntheses. In addition, since
quantum dots usually are synthesized in organic solvents and thus
capped with hydrophobic ligands, facile surface/interface
chemistries are required to render I--III-VI quantum dots
water-dispersible and also to provide functional groups for
subsequent bio-conjugation with biological moieties. The disclosed
composites and compositions address this need. Additionally, the
composites disclosed herein possess several merits including dual
imaging properties, high (Mn+Fe) recovery, excellent stability in
aqueous solutions with a wide pH/ionic-strength range and
physiological media, minimal cytotoxicity, the ability to tune
photoluminescence by surface doping, and cell targeting
capabilities before and after bio-conjugation. Without being
limited to a particular theory, it is currently believed that the
high recovery of the composites is attributed to the efficient
wrapping of the composite cores by the aliphatic tails of the
zwitterionic polymeric coating, and that the colloidal stability,
and minimal cytotoxicity for cell targeting results from the nature
of the incorporated zwitterionic groups.
Additionally, the composites can be used for cell imaging and the
photoluminescence tunability of the composites from the visible to
near infrared (NIR) range contributes to their utility in in vivo
diagnosis. The fluorescence imaging data obtained from the
composites (e.g., NIR and magnetic resonance imaging) may also be
used to provide complimentary information on tumor biology and thus
enhance diagnosis accuracy.
III. Composites and Compositions
Disclosed herein are embodiments of composites and compositions
thereof, wherein the composites comprise a core having a
nanoparticle, quantum dot, or combination thereof and a polymeric
coating that encapsulates the core. In exemplary embodiments, the
composites comprise magnetofluorescent cores and a zwitterionic
polymeric coating suitable for promoting biocompatibility and
coupling to biomolecules. In particular disclosed embodiments, the
magnetofluorescent core comprises at least one quantum dot and at
least one magnetic nanoparticle.
The zwitterionic polymer coating, which comprises a polymeric
backbone from which zwitterionic side chains extend, can be used to
encapsulate the core of the composite, such as by forming a
polymeric layer that surrounds a composition of magnetic
nanoparticles, quantum dots, or a combination thereof. In
particular disclosed embodiments, the zwitterionic polymeric
coating comprises a polymer having a hydrophobic portion and a
hydrophilic portion. The hydrophobic portion can be oriented so
that it interacts with the core comprising the magnetic
nanoparticles, quantum dots, or combination thereof to form a
micellar core. The hydrophilic portion can be oriented so that it
extends from the micellar core to promote solubility and/or
biomolecular conjugation.
In some embodiments, the zwitterionic polymeric coating can
comprise a polymer having a pH-responsive group, which can comprise
a combination of a functional group positioned on the polymeric
chain of the polymer, which comprises a tertiary amine, and a
functional group positioned on the polymeric chain of the polymer,
which comprises a carboxyl group. The combination of these two
functional groups can provide a weak zwitterionic moiety. In such a
weak zwitterion, the free proton from the carboxyl group can be
transferred to the tertiary amine without affecting the neutral
charge of the whole pair. In some embodiments, the pK.sub.a of the
carboxyl group of the weak zwitterionic moiety is around 5.0-6.0,
and can be protonated in acidic endosomes and thus lead to osmotic
swelling and endosome rupture benefitting the ensodomal escape of
micelles. In yet additional embodiments, the zwitterionic polymer
can comprise carboxybetaine groups that can also absorb protons in
acidic conditions for endosomal escape. In some embodiments, the
molar ratio of functional groups present on the zwitterionic
polymeric coating (e.g., carboxybetaine groups, sulfobetaine
groups, pH responsive groups, and thiol-containing groups) can be
modified to provide a desired reactivity of the micelle formed with
the polymeric coating and the core.
In particular disclosed embodiments, the zwitterionic polymeric
coating can comprise a polymer having a structure meeting Formula I
below.
##STR00001## With reference to Formula I, each R.sup.1 can provide
the hydrophobic portion of the polymer and independently can be
selected from hydrogen or aliphatic; each R.sup.2 independently can
be --C(O)Z, wherein Z can be selected from hydroxyl, ether, amine,
thiol, or thioether; each R.sup.3 can (alone or in combination with
R.sup.2) provide the hydrophilic portion of the polymer and
independently can be selected from amide-aliphatic-amine (e.g.,
--CO(NR.sup.b)C1-C10alkylN(R.sup.b).sub.2, wherein each R.sup.b
independently can be selected from hydrogen, aliphatic,
heteroaliphatic, aryl, or heteroaryl),
amide-aliphatic-amine-aliphatic-carboxylate (e.g.,
--CO(NR.sup.b)C1-C10alkyl
[N(R.sup.b).sub.2].sup.+C1-C10alkyl-CO.sub.2.sup.-, wherein each
R.sup.b independently can be selected from hydrogen, aliphatic,
heteroaliphatic, aryl, or heteroaryl),
amide-aliphatic-amine-aliphatic-sulfonate (e.g.,
--CO(NR.sup.b)C1-C10alkyl
[N(R.sup.b).sub.2].sup.+C1-C10alkyl-SO.sub.3.sup.-, wherein each
R.sup.b independently can be selected from hydrogen, aliphatic,
heteroaliphatic, aryl, or heteroaryl), or amide-aliphatic-thiol
(e.g., --CO(NR.sup.b)C1-C10alkylSH, wherein R.sup.b can be selected
from hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl); p
can be an integer selected from zero to 5; q can be an integer
selected from zero or 1; and each of a and b independently can be
an integer selected from 1 to 200.
In some embodiments, the zwitterionic polymeric coating can
comprise a polymer having a structure satisfying any one of
Formulas II-VI below.
##STR00002## ##STR00003##
With reference to Formulas II-VI, each R.sup.1 independently can be
selected from hydrogen or aliphatic; each X.sup.1, X.sup.2,
X.sup.3, X.sup.4, and X.sup.5 independently can be selected from
NR.sup.b, N(R.sup.b).sub.2.sup.+, oxygen, or sulfur, wherein each
R.sup.b independently can be selected from hydrogen, aliphatic,
heteroaliphatic, aryl, or heteroaryl; each A.sup.1, A.sup.2,
A.sup.3, A.sup.4, and A.sup.5 independently can be selected from
aliphatic or heteroaliphatic; each Y.sup.1, Y.sup.2, and Y.sup.3
independently can be selected from amine, thiol, carboxylate or
sulfonate; each Z independently can be selected from hydroxyl,
ether, amine, thiol, or thioether; n can be an integer selected
from 1 to 200; each m can be an integer selected from 0 to 3, such
as 0, 1, 2, or 3; each p independently can be an integer selected
from zero to 5; a can be an integer selected from 1 to 200, such as
1 to 100 or 5 to 50; and b and c independently can be an integer
selected from 0 to 200, such as 1 to 100 or 5 to 50.
In particular disclosed embodiments, each R.sup.1 independently can
be selected from hydrogen, alkyl, alkenyl, or alkynyl; each
X.sup.1, X.sup.2, X.sup.3, X.sup.4, and X.sup.5 independently can
be selected from NR.sup.b, N(R.sup.b).sub.2.sup.+, oxygen, or
sulfur, wherein each R.sup.b independently can be selected from
hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, phenyl, naphthyl, or pyridinyl; each A.sup.1,
A.sup.2, A.sup.3, A.sup.4, and A.sup.5 independently can be
selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
or heteroalkynyl; each Y.sup.1 and Y.sup.2 independently can be
selected from carboxylate or sulfonate; Y.sup.3 can be SH; each Z
independently can be selected from hydroxyl, thiol, or NH.sub.2;
and m is 1.
In yet additional embodiments, each R.sup.1 independently can be
selected from hydrogen or C1-C20 alkyl (such as C1-C16 alkyl, or
C1-C10 alkyl); each X.sup.1, X.sup.2, X.sup.3, X.sup.4, and X.sup.5
independently can be selected from NH or NMe.sub.2.sup.+; each
A.sup.1, A.sup.2, A.sup.3, A.sup.4, and A.sup.5 independently can
be selected from C1-C20 alkyl (such as C1-C10 alkyl or C1-C5 alkyl,
or C1-C3 alkyl); each Y.sup.1 and Y.sup.2 independently can be
selected from carboxylate or sulfonate; Y.sup.3 can be SH; and each
Z independently can be hydroxyl. In exemplary embodiments, the
zwitterionic polymer coating can have any one of the structures
below.
##STR00004## ##STR00005##
The composites disclosed herein can comprise a core that includes a
nanoparticle, a quantum dot, or a combination thereof. In
particular embodiments, the core comprises a magnetic nanoparticle,
a quantum dot, or a combination thereof. In yet additional
embodiments, the core comprises a magnetic nanoparticle and a
quantum dot. In such embodiments, the core is considered
magnetofluorescent. In particular disclosed embodiments, the
composite comprises MnFe.sub.2O.sub.4 magnetic nanoparticles,
CuInS.sub.2 quantum dots, AgInS.sub.2 quantum dots, or any
combination thereof.
In some embodiments, the nanoparticles are magnetic and can be
nanoparticles comprising a metal selected from Mn, Fe, Ni, Co, or
combinations thereof. In particular disclosed embodiments, the
magnetic nanoparticle comprises a metal and/or a metal oxide, such
as an iron oxide, a nickel oxide, a cobalt oxide, a manganese
oxide, or a combination thereof. Exemplary magnetic nanoparticles
include, but are not limited to MnFe.sub.2O.sub.4, Fe.sub.3O.sub.4,
CoFe.sub.2O.sub.4, FePt, or a combination thereof. In some
embodiments, the nanoparticles, quantum dots, or both can have core
sizes of greater than 1 nm to 10 nm, such as 1 nm to 8 nm, or 1 nm
to 5 nm.
Suitable quantum dots that can be used in the composites disclosed
herein include, but are not limited to, tertiary or ternary quantum
dots, such as AgInS.sub.2, Ag(In,Ga)Se.sub.2, Ag(Zn,Sn)Se.sub.2,
Ag(Zn,Sn)S.sub.2, AgIn(Se,S).sub.2, AgZn(Se,S).sub.2,
AgSn(Se,S).sub.2, and Ag(Zn,Sn)(Se,S).sub.2ZnSSe, ZnSeTe, ZnSTe,
ScSTe, HgSSe, HgSeTe, HgSTe, ZnHgS, ZnHgSe, ZnHgTe, ZnHgSSe,
ZnHgSeTe, InGaAs, GaAlAs, InGaN, CuInS.sub.2, Cu(In,Ga)Se.sub.2,
Cu(Zn,Sn)Se.sub.2, Cu(Zn,Sn)S.sub.2, CuIn(Se,S).sub.2,
CuZn(Se,S).sub.2, CuSn(Se,S).sub.2, and Cu(Zn,Sn)(Se,S).sub.2. In
some embodiments, the quantum dots used in the composites are
quantum dots. Exemplary quantum dots include, but are not limited
to, CuInS.sub.2 quantum dots and AgInS.sub.2 quantum dots. The
quantum dot can be a core quantum dot or a core/shell quantum dot.
In core/shell embodiments, the quantum dot can be selected from any
quantum dot described herein and can further comprise a shell made
of a binary material selected from, but not limited to, CdS, CdSe,
CdTe, GaAs, InAs, InN, InP, InSb, PbS, PbSe, PbTe, ZnS, ZnSe, or
ZnTe; an ion species, such as a halide (e.g., bromide, chloride,
fluoride, or iodide); or a combination thereof. In exemplary
embodiments, ZnS or chloride is selected as the shell component.
Exemplary embodiments of core/shell quantum dots include, but are
not limited to, CuInS.sub.2/ZnS quantum dots and AgInS.sub.2/ZnS
quantum dots. Core and core/shell quantum dots can be used in
combination with nanoparticles, particularly nanoparticles, to form
an overall core used in the composites disclosed herein.
The core of the composite also can be doped with one or more
metals. In particular disclosed embodiments, quantum dots (core
and/or core/shell quantum dots) and/or nanoparticles (e.g.,
magnetic nanoparticles) can be doped with a metal selected from Cu,
Ag, Au, Mn, Zn, or combinations thereof. Doping the cores of the
disclosed composites provides the ability to tune the optical
properties and material characteristics of the cores and also can
prolong the photoluminescence lifetime of the composites disclosed
herein. Photoluminescence tuning of the composites can be used
avoid cross-talk with other existing dyes in bioimaging/sensing,
and also provides design flexibility for optoelectronic devices,
such as light-emitting diodes (LEDs) with different color
emissions. Like other composites disclosed herein, the doped cores
can be encapsulated with the zwitterionic polymeric coatings
disclosed herein to form composites. The doped composites can be
used as photoluminescent probes in cellular imaging and tissue
imaging.
Also disclosed herein are compositions comprising the composites
described above and one or more biomolecules. In some embodiments,
the biomolecules of the composition can be the same. In yet other
embodiments, multiple different biomolecules can be used.
Biomolecules can be selected from targeting ligands, antibodies,
nucleic acids, therapeutic molecules, or a peptide having
biofunctionality, or combinations thereof. In exemplary
embodiments, the biomolecules can be selected from chlorotoxin,
avidin, biotin, folic acid, arginylglycylaspartic acid, or
combinations thereof.
The biomolecules can be chemically conjugated to the composites,
such as electrostatically and/or covalently. In some embodiments,
biomolecules can be covalently bound to functional groups on the
composite, such as carboxylate groups of the zwitterionic polymer
coating. In some embodiments, the composites can comprise a mixture
of carboxylate and sulfonate functional groups to facilitate
conjugation to biomolecules through the carboxylate groups and also
to maintain the zwitterionic nature of the polymer using sulfonate
groups, which do not react with the biomolecules. In some
embodiments, the disclosed compositions can be used to determine
and demonstrate the biomedical uses of the composites and
compositions, such as by performing cellular/tissue studies to
investigate cytotoxicity of the composites and/or compositions and
specific cellular binding/uptake of conjugated composites by cells,
such as tumor cells.
In some embodiments, drugs, such as chemotherapy drugs (e.g.,
doxorubicin, daunorubicin, epirubicin, idarubicin, or the like) can
be loaded into the cores of micelles for therapeutic drug delivery
applications. Once exposed to physiological conditions (e.g.,
circulation environments), drugs encapsulated in micelles usually
are released within the first several hours, after which their
concentrations reach a saturation plateau. The burst release should
be minimal before micelles are internalized into target cells, but
efficient after they are in target cells. To prevent premature drug
leakage, thiol groups can be incorporated into the zwitterionic
polymer to form disulfide cross-linkages in oxidative conditions
and thereby form a cross-linked layer of the zwitterionic polymeric
coating. FIG. 33 illustrates an exemplary embodiment wherein a
cross-linked layer 3300 is formed by thiol groups of a polymeric
coating backbone. The presence to thiol groups can help different
polymer backbones to cross link with each other and form a shell on
the surface. This shell is stable under physiological conditions
minimizing drug leakage, but sensitive to and degradable in
intracellular reductive-microenvironments. As illustrated in FIG.
33, the cross-linked layer can encapsulate quantum dots 3302 and
drug molecules 3304. The devised shell is designed to help micelles
to achieve more drug release inside cells.
The composites disclosed herein can be characterized using a
variety of techniques. In some embodiments, Fourier transform
infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR)
spectroscopy can be used to characterize the composites. In yet
additional embodiments, the composites can be characterized based
on their particle composition and size, iron content recovery
rates, optical properties, colloidal stability, and magnetic
relaxivity in harsh conditions using transmission electron
microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy,
dynamic light scattering (DLS), UV-Vis spectroscopy, fluorescence
spectroscopy, magnetic resonance spectroscopy, and combinations
thereof.
IV. Methods of Making Composite Components, Composites, and
Compositions
Described herein are embodiments of methods for making the
composite components described above, the composites themselves,
and compositions comprising the composites. Methods for making
composite components, such as the zwitterionic polymer coating and
the quantum dots that can be used in the magnetofluorescent core
also are described below.
In some embodiments of making the zwitterionic polymers described
herein, the methods comprise converting a polymer precursor to a
polymeric intermediate, which can then be reacted with suitable
reagents that provide the zwitterionic portion of the polymer.
##STR00006## As illustrated in Scheme 1, polymeric precursor 100
can be converted to polymeric intermediate 102 by addition of an
appropriate nucleophile to polymeric precursor 100, which
ring-opens to provide polymeric intermediate 102. With respect to
Scheme 1, each R.sup.1, R.sup.2, X.sup.2, A.sup.1, m a, and p can
be as recited above; Z.sup.a can be selected from oxygen or
NR.sup.b; and each R.sup.4 independently can be selected from
hydrogen or aliphatic. In particular embodiments, a method as
illustrated in Scheme 2 can be used to produce polymeric
intermediate 202 from polymeric precursor 200. In some embodiments,
the zwitterionic polymer coating can comprise functional groups
suitable for forming disulfide cross-linkages within the polymeric
coating to thereby form a cross-linked layer of the polymer (FIG.
33). In such embodiments, the disulfide-containing polymers can be
made by reacting a polymeric precursor with a disulfide-containing
reagent, such as 2-(2-pyridyldithio)ethylamine. The disulfide bond
of the disulfide-containing reagent can be reduced to a thiol using
excess dithiothreitol (DTT). The crosslinking between thiol groups
of the polymeric coating can be conducted under aqueous oxidative
conditions during the removal of DTT from micelles by dialysis
against PBS buffer.
##STR00007##
The polymeric intermediate can then be converted to a zwitterionic
polymer 300 by reacting polymeric intermediate 102 with one or more
different electrophiles capable of providing a carboxylate moiety,
a sulfonate moiety, or a combination thereof, as illustrated in
Scheme 3 below. In some embodiments, a cyclic lactone and/or a
cyclic sulfone can be used as the electrophile component.
##STR00008##
An exemplary method of producing a zwitterionic polymer is
illustrated below in Scheme 4. As illustrated in Scheme 4,
polymeric intermediate 202 is reacted with .beta.-propiolactone and
1,3-propanesultone in one step to provide the carboxylate- and
sulfonate-containing zwitterionic polymer 400. This method can be
used to produce such compounds in one step without having to
utilize multiple steps to attach both the carboxylate and the
sulfonate moieties. The resulting zwitterionic polymer possesses
carboxybetaine groups, sulfobetaine groups, and hydrophobic groups
on their backbones. Compared to sulfobetaine, carboxybetaine groups
are not only zwitterions but also supply carboxyl heads for
bio-conjugation with conventional EDC/NHS cross-linkers. The
sulfobetaine groups are insensitive to regular bio-crosslinking
reactions and thus keep the zwitterionic property of the
amphiphiles during conjugation. Although simple carboxyl groups are
presented on the polymer backbone, carboxybetaine in the polymeric
coating can further increase the biomolecule loading capability of
composites through bio-crosslinking. Moreover, carboxybetaine
branches can be provided that are similar to or substantially
similar to the length of the sulfobetaine branches and therefore
are easy to access for bio-conjugation.
##STR00009##
In yet additional embodiments, the zwitterionic polymeric coating
can be made by first making a functionalized zwitterionic
intermediate 500 and then reacting the functionalized zwitterionic
intermediate 500 with a polymeric precursor 100, such as is
illustrated in Scheme 5.
##STR00010## With respect to Scheme 5, each of R.sup.1, R.sup.2,
R.sup.4, X.sup.1, X.sup.2, A.sup.1, Z.sup.a, m, a, and p can be as
recited above. A representative method of making the zwitterionic
polymer in this fashion is illustrated in Scheme 6 below.
##STR00011##
To produce the composites, the zwitterionic polymeric coating
together with hydrophobic magnetic nanoparticles, quantum dots, or
a combination thereof can be dissolved in organic solvents and then
dispersed into water under sonication. FIG. 32 provides an
exemplary schematic of a representative method of making micelles
comprising the polymeric coatings disclosed herein. As illustrated
in FIG. 32, an organic solution 3200 comprising a polymer as
disclosed herein, one or more quantum dots, nanoparticles, a drug,
or any combination thereof is mixed with water and undergoes
sonication using a sonicator 3202. After sonication the micelles
begin to form (3204) and vacuum removal of the organic solvents
provides the formed micelles (3206). After dispersion, organic
solvents can be removed by vacuum under rotary evaporation. In the
dispersion and vacuum, hydrophobic alkyl tails in the amphiphiles
interact with hydrophobic nanoparticles, quantum dots, or both to
form the stable micellar cores, and carboxylate and sulfonate
groups are exposed to water.
In some embodiments, the quantum dots used in the composites
disclosed herein can be made using a thermal decomposition method.
The thermal decomposition method can be easily scaled up to a
commercial scale for quantum dot production as compared to
conventional hot-injection methods. In some embodiments, a quantum
dot precursor is used in the thermal decomposition method. In
comparison to hot-injection methods, the present method does not
require multiple washing and dryings steps or the use of toxic
sodium diethyldithiocarbamate to produce the quantum dot precursor.
Instead, the present methods use metal precursors and non-toxic
ligands to produce the quantum dots in situ. No complex precursors,
external sulfur sources, or other solvents are needed. Also, all
chemicals used in the methods are commercially available.
The thermal decomposition methods described herein can comprise
combining a Group 11 metal precursor, a Group 13 metal precursor,
and a ligand in a thiol-containing solvent. In some embodiments,
the Group 11 metal precursor is selected from a silver precursor or
a copper precursor. In some embodiments, the Group 13 metal
precursor is an indium precursor. In particular disclosed
embodiments, silver acetate or copper acetate can be used as the
Group 11 metal precursor and indium acetate can be used as the
Group 13 metal precursor. Suitable ligands include, but are not
limited to fatty acids, such as oleic acid, palmitoleic acid,
linoleic acid, and the like. In some embodiments, the
thiol-containing solvent is dodecanethiol, octadecanethiol,
octanethiol, decanethiol, or combinations thereof.
The Group 11 metal precursor, the Group 13 metal precursor, and the
ligand are combined in the thiol-containing solvent and are heated
at a temperature suitable to facilitate thermal decomposition of
the precursors to thereby form the desired quantum dots, such as
quantum dots. In some embodiments, the temperature ranges from
150.degree. C. to 200.degree. C., such as 160.degree. C. to
190.degree. C., or 170.degree. C. to 180.degree. C. In particular
disclosed embodiments, the reaction mixture of the Group 11 metal
precursor, the Group 13 metal precursor, the ligand, and the
solvent is heated at 170.degree. C.
In some embodiments, the methods can use a particular ratio of
metal precursors to provide the disclosed quantum dots. In some
embodiments, a Group 11:Group 13 metal precursor molar ratio
ranging from 1:1 to 1:5 can be used, such as 1:1 to 1:4, or 1:1 to
1:3, or 1:1 to 1:2. In exemplary embodiments, an Ag:In molar ratio
of 1:2 or a Cu:In molar ratio of 1:2 can be used.
In some embodiments, the method can further comprise growing a
shell on the quantum dot surface to enhance quantum yield. To
enhance the quantum dot quantum yield (QY), a ZnS shell can grown
on the quantum dot surface to form quantum dots having increased
quantum yield as compared to quantum dots without a shell.
In an exemplary embodiment of the thermal decomposition methods
disclosed herein, which is described solely by way of example,
indium acetate and silver acetate or copper acetate can be reacted
with a sulfur source, such as dodecanethiol, to form intermediate
compounds of Ag(SC.sub.12H.sub.25).sub.x or
Cu(SC.sub.12H.sub.25).sub.x and In(SC.sub.12H.sub.25).sub.x upon
heating and are dissolved in dodecanethiol. As the reaction
temperature is raised to a temperature ranging from 150.degree. C.
to 200.degree. C. (e.g., 160.degree. C. to 190.degree. C., or
170.degree. C. to 180.degree. C.) the intermediate compounds can
act as precursors and further decompose to form Ag--S or Cu--S and
In--S to form Ag--In--S or Cu--In--S particles. Without being
limited to a particular theory, it is currently believed that a 1:2
molar ratio of Cu:In or Ag:In can be used to circumvent the
possibility of producing Ag.sub.2S or Cu.sub.2S particles. To
further enhance the quantum yield, the AIS or CIS quantum dots can
be passivated with a shell, such as a ZnS shell to form AIS/ZnS or
CIS/ZnS quantum dots. In some embodiments, the core/shell quantum
dots exhibited a significant quantum yield up to 41% but with a
blue shift, as demonstrated in FIG. 1.
Embodiments of doped composites can be made using new synthetic
methods disclosed herein that are reproducible and avoid multiple
washing/drying steps and complex synthetic reactions. In particular
disclosed embodiments, the methods use dopant precursors that are
commercially available and relatively safe to use (e.g., no glove
box is needed in preparing these precursors, and no highly
restricted handling or disposal is required). In some embodiments,
the methods described above for making the composites can be
further modified to include a step whereby dopant precursors, such
as CuI, Mn(acetate).sub.2, Zn(acetate).sub.2, AgNO.sub.3,
Au(acetate).sub.3, and the like, are added to composite solutions
to provide surface-doped composites. In yet additional embodiments,
doped composites comprising a core passivated with a shell can be
made by coating and/or etching the shell components onto a doped
core composition formed using a dopant precursor and a composite
solution. Thus, in particular disclosed embodiments, a surface
doping method is used, in contrast to a homogenous doping technique
where the dopant is merely added to the precursors used to make the
composite. The amount of dopant added can range from greater than 0
mol % to 15 mol %, such as from greater than 0 mol % to 10 mol %,
or 3 mol % to 10 mol %, or 6 mol % to 10 mol %. In some
representative embodiments, 3.33 mol %, 6.67 mol % and 10 mol % of
the dopant can be added. In particular disclosed embodiments, a
dopant precursor can be dissolved in a suitable solvent and then
added dropwise to a solution comprising the un-doped composite. The
composite solution can be heated to temperatures ranging from
120.degree. C. to 200.degree. C. or higher prior to addition of the
dopant. After the dopant is added, the resulting reaction mixture
can be cooled to ambient temperature. Methods for making doped
core/shell composites can comprise preparing a doped composite as
described above and then growing the shell on the doped core by
adding selected shell precursors to a solution comprising the doped
composite. In particular disclosed embodiments, the amount of the
shell precursor added to the solution typically is an amount that
would provide a sufficient number of shell atoms absorbed on to the
surface of the doped composite; thus, in some embodiments, the
amount can be determined based on the diameter of the doped
composite. As an exemplary embodiment, doped core/shell composites
can be obtained by preparing a shell precursor solution having a
fixed concentration of the shell precursor, such as
Zn(acetate).sub.2, and then injecting a selected volume of the
shell precursor solution (e.g., 0.5 mL) into a reactor comprising
the doped composite. In some embodiments, at least 1 to 20
injections, such as 1 to 15 injections, or 2 to 10 injections, or 4
to 9 injections of the shell precursor solution can be added. After
each injection, the quantum yield of the composites can be assessed
to provide a curve of quantum yield as a function of injection
time. This curve can be used to obtain suitable injection times and
the total amount of the precursor solution to be added to provide
the desired core/shell doped composites.
The doping methods described above can be used to provide doped
composites that exhibit high quantum yields (e.g., 50% or higher,
such as 60% or higher) and prolonged photoluminescent lifetimes
(e.g., 300 ns or more, such as 500 ns or more). The optical
properties and material characteristics of the doped composites can
be characterized using photoluminescence spectroscopy, UV-Vis
spectroscopy, X-ray diffraction (XRD), transmission electron
microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy,
time-resolved photoluminescence spectroscopy, or combinations
thereof.
V. Methods of Using Composites and Compositions
The composites and compositions described herein can be used for
biomedical imaging, biosensing, and therapeutic treatment. In some
embodiments, the composites and compositions can be used for cell-
or tissue-based diagnosis and therapy. For example, the composites
and compositions can be used as contrast agents and/or probes for
imaging cells, tumors, and/or detecting endogenous targets
expressed in cells. In some embodiments, the compositions can
comprise a composite and a biomolecule having therapeutic activity
and/or a therapeutic drug and therefore the compositions can be
used to treat, prevent, and/or ameliorate a disease or disorder.
The disclosed composites and compositions are non-toxic, stable,
and aqueous-soluble, and thus are suited for imaging of subjects
both in vitro and in vivo as well as for drug delivery to a
subject. In some embodiments, the composites and/or composite
components (e.g., cores and/or zwitterionic polymer) can be used as
imaging probes for flow cytometry, cell and tissue staining, cell
tracking, Western blotting, in vivo imaging, and the like. In yet
additional embodiments, the composites can be used as sensing
probes. An exemplary sensing probe is illustrated in FIG. 43.
According to the embodiment illustrated in FIG. 43, a sensing probe
4300 comprises an absorbent portion 4302, a capturing antibody
portion 4304, a membrane (e.g., a nanocellulose membrane) 4306, a
composite portion 4308, which comprising one or more of the
composites disclosed herein, and a sample application portion 4310.
Sensing probes like that illustrated in FIG. 43 can be used to
detect the presence of a particular analyte of interest. When such
sensors are used, the photoluminescence produced by the composite
portion can be used as a signal under continuous excitation. As the
composites exhibit long photoluminescence lifetimes, the
time-resolved photoluminescence of these composites also can be
used as signals. In such embodiments, the time-resolved
photoluminescence signals are produced by exciting the composites
with a pulse light source; after the pulse excitation light is
switched off, the composites will still emit fluorescence for
several hundred nanoseconds or longer. Such a time-resolved signal
can avoid the interference of any background
(autofluorescence).
In particular disclosed embodiments, the disclosed composites are
combined with a biomolecule to produce a composition. The
biomolecule can be coupled to the composite chemically, such as
through a covalent or electrostatic bond. In some embodiments, the
biomolecule can be covalently coupled to the composite using
conjugation reagents and methods, such as by maleimide-thiol
reaction chemistry, NHS-ester reaction chemistry, amino acid
couplings, and the like. The coupling of the composite and the
biomolecule can be evaluated and confirmed using FT-IR
spectroscopy.
After conjugation of the composite and the biomolecule to form the
composition, it can be administered to a subject or it can be
exposed to a sample in vitro. The composition can be administered
using any suitable administration route, such as oral
administration, injection, topical administration, inhalation, or a
combination thereof. In particular disclosed embodiments, the
composition can further comprise a pharmaceutically acceptable
carrier, such as water or other pharmaceutically acceptable
solvent. In some embodiments, the composition can be administered
as a solution, a dispersion, a tablet, or a capsule.
In some embodiments, the doped composites disclosed herein can be
in various biomedical applications, such as cell targeting and
imaging applications. In particular disclosed embodiments, the
doped composites comprise a zwitterionic polymeric coating
disclosed herein. These doped composites can be conjugated to
targeting moieties, such as antibodies, haptens, peptides, a member
of a specific binding pair, and other suitable targeting
biomolecules. In particular disclosed embodiments, the doped
composites can be coupled to peptides using peptide coupling
reagents, such as, but are not limited to
2-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium
hexafluorophosphate,
2-(1H-benzotriazol-1-yl)-N,N,N',N'-hexafluorophosphate,
2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium
hexafluorophosphate, 1-hydroxybenzotriazole,
dicyclohexylcarbodiimide, diisopropylcarbodiimide,
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide-HCl,
benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium
hexafluorophosphate,
benzotriazol-1-yloxy-tripyrrolidino-phosphonium
hexafluorophosphate, bromo-tripyrrolidino-phosphonium
hexafluorophosphate, and the like, or a combination thereof. These
peptide coupling reagents also can be used for other composites
disclosed herein (that is, non-doped composites). In particular
disclosed embodiments, the doped composites can be coupled to an
RGD peptide for targeting tumors and/or mutations in brain cells,
kidney cells, liver cells, breast tissue cells, and the like. In
yet additional embodiments, the doped composites (and other
composites disclosed herein) can be combined with therapeutics
and/or image contrast agents to deliver drugs to particular targets
within a subject and/or to provide additional imaging of such
targets.
VI. Overview of Several Embodiments
Some embodiments disclosed herein concern composites comprising a
core comprising one or more magnetic nanoparticles, one or more
quantum dots, or a combination thereof; and a zwitterionic
polymeric coating comprising a zwitterionic polymer having a
structure satisfying a formula
##STR00012## wherein each R.sup.1 independently is selected from
hydrogen or aliphatic; each R.sup.2 is --C(O)Z, wherein Z is
selected from hydroxyl, ether, amine, thiol, or thioether; each
R.sup.3 independently is selected from amide-aliphatic-amine,
amide-aliphatic-amine-aliphatic-carboxylate,
amide-aliphatic-amine-aliphatic-sulfonate, or
amide-aliphatic-thiol; p is an integer selected from zero to 5; q
is an integer selected from zero or 1; and each of a and b
independently is an integer selected from 1 to 200. In some
embodiments, the zwitterionic polymeric coating comprises a
zwitterionic polymer having a structure satisfying a formula
selected from
##STR00013## wherein each R.sup.1 independently is selected from
hydrogen or aliphatic; each of X.sup.1, X.sup.2, X.sup.3, X.sup.4,
and X.sup.5 independently is selected from NR.sup.b,
N(R.sup.b).sub.2.sup.+, oxygen, or sulfur, wherein each R.sup.b
independently is selected from hydrogen, aliphatic,
heteroaliphatic, aryl, or heteroaryl; each of A.sup.1, A.sup.2,
A.sup.3, A.sup.4, and A.sup.5 independently is selected from
aliphatic or heteroaliphatic; each of Y.sup.1, Y.sup.2, and Y.sup.3
independently is selected from amine, thiol, carboxylate or
sulfonate; each Z independently is selected from hydroxyl, ether,
amine, thiol, or thioether; n is an integer selected from 1 to 200;
each m is an integer selected from 0 to 3; each p independently is
an integer selected from zero to 5; each of a, b, and c
independently is an integer selected from 1 to 200.
In any or all of the above embodiments, each R.sup.1 independently
is selected from hydrogen, alkyl, alkenyl, or alkynyl; each of
X.sup.1, X.sup.2, X.sup.3, and X.sup.5 independently is selected
from NR.sup.b, N(R.sup.b).sub.2.sup.+, oxygen, or sulfur, wherein
each R.sup.b independently is selected from hydrogen, alkyl,
alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,
phenyl, naphthyl, or pyridinyl; each of A.sup.1, A.sup.2, A.sup.3,
A.sup.4, and A.sup.5 independently is selected from alkyl, alkenyl,
alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl; each of
Y.sup.1 and Y.sup.2 independently is selected from carboxylate or
sulfonate and Y.sup.3 is thiol; each Z is hydroxyl; and m is 1.
In any or all of the above embodiments, the zwitterionic polymeric
coating comprises a zwitterionic polymer have a structure
##STR00014## ##STR00015##
In any or all of the above embodiments, the core comprises at least
one magnetic nanoparticle and at least one quantum dot.
In any or all of the above embodiments, the core is doped with a
metal selected from Cu, Ag, Au, Mn, Zn, or combinations thereof. In
some embodiments, the core is doped with greater than 0 mol % to 10
mol % of the metal.
In any or all of the above embodiments, the core is a quantum dot
core and the quantum dot core comprises a ZnS shell. In some
embodiments, the quantum dot core exhibits a quantum yield of
greater than 50%. In any or both of these embodiments, the quantum
dot core exhibits a photoluminescence lifetime ranging from 300 to
500 nanoseconds.
In any or all of the above embodiments, the quantum dot is a
quantum dot.
In any or all of the above embodiments, wherein the quantum dot
comprises copper or silver.
In any or all of the above embodiments, the quantum dot is an
AgInS.sub.2 quantum dot or a CuInS.sub.2 quantum dot.
In any or all of the above embodiments, the quantum dot further
comprises a ZnS shell.
In any or all of the above embodiments, the magnetic nanoparticle
comprises Fe.
In any or all of the above embodiments, wherein the magnetic
nanoparticle is MnFe.sub.2O.sub.4, Fe.sub.3O.sub.4,
CoFe.sub.2O.sub.4, FePt or a combination thereof.
In any or all of the above embodiments, the core comprises a
combination of MnFe.sub.2O.sub.4 nanoparticles and an AgInS.sub.2
quantum dot, a CuInS.sub.2 quantum dot, or a combination
thereof.
Also disclosed herein are embodiments of composites comprising: a
core comprising a combination of MnFe.sub.2O.sub.4 nanoparticles
and CuInS.sub.2 quantum dots, AgInS.sub.2 quantum dots, or both;
and a zwitterionic polymeric coating comprising a zwitterionic
polymer having a structure selected from
##STR00016## ##STR00017##
In any or all of the above embodiments, the core further comprises
a dopant, a shell, or both. In some embodiments, the dopant is a
metal selected from Cu, Ag, Au, Mn, Zn, or combinations thereof. In
some or both of these embodiments, the shell is a ZnS or chloride
shell.
Also disclosed herein are embodiments of compositions, comprising:
a composite having a core comprising one or more magnetic
nanoparticles, one or more quantum dots, or a combination thereof
and a zwitterionic polymeric coating comprising a zwitterionic
polymer having a structure satisfying a formula
##STR00018##
wherein each R.sup.1 independently is selected from hydrogen or
aliphatic; each R.sup.2 is --C(O)Z, wherein Z is selected from
hydroxyl, ether, amine, thiol, or thioether; each R.sup.3
independently is selected from amide-aliphatic-amine,
amide-aliphatic-amine-aliphatic-carboxylate,
amide-aliphatic-amine-aliphatic-sulfonate, or
amide-aliphatic-thiol; p is an integer selected from zero to 5; q
is an integer selected from zero or 1; and each of a and b
independently is an integer selected from 1 to 200; and a
biomolecule, a drug, or a combination thereof. In some embodiments,
the core further comprises a dopant, a shell, or both. In some or
both of these embodiments, the dopant is a metal selected from Cu,
Ag, Au, Mn, Zn, or combinations thereof.
In any or all of the above embodiments, the shell is a ZnS or a
chloride shell.
In any or all of the above embodiments, the biomolecule is
conjugated to the composite.
In any or all of the above embodiments, the biomolecule is
chemically conjugated to the composite through the zwitterionic
polymeric coating.
In any or all of the above embodiments, the biomolecule is
chemically conjugated to the composite through a carboxylate group
of the zwitterionic polymeric coating. In some embodiments, the
carboxylate group of the zwitterionic polymeric coating is further
chemically bound to a linker, wherein the linker is also chemically
bound to the biomolecule.
In any or all of the above embodiments, the biomolecule is selected
from chlorotoxin, avidin, biotin, folic acid, arginylglycylaspartic
acid, or combinations thereof.
In any or all of the above embodiments, the drug is encapsulated
within the zwitterionic polymeric coating.
In any or all of the above embodiments, the drug is doxorubicin,
daunorubicin, epirubicin, idarubicin, or a combination thereof.
Also disclosed herein are embodiments of methods for making a
composite, comprising: combining, to form a mixture, a solution
comprising one or more magnetic nanoparticles, one or more quantum
dots, or a combination thereof with a solution of a zwitterionic
polymer having a structure satisfying a formula
##STR00019## wherein each R.sup.1 independently is selected from
hydrogen or aliphatic; each R.sup.2 is --C(O)Z, wherein Z is
selected from hydroxyl, ether, amine, thiol, or thioether; each
R.sup.3 independently is selected from amide-aliphatic-amine,
amide-aliphatic-amine-aliphatic-carboxylate,
amide-aliphatic-amine-aliphatic-sulfonate, or
amide-aliphatic-thiol; p is an integer selected from zero to 5; q
is an integer selected from zero or 1; and each of a and b
independently is an integer selected from 1 to 200; dispersing the
mixture into water using sonication to form a dispersed
composition; and isolating the composite from the dispersed
composition.
In any or all of the above embodiments, the method further
comprises centrifuging the dispersed composition to remove
non-composite by-products.
Also disclosed herein are embodiments of methods for making a
quantum dot, comprising: combining a Group 11 metal precursor with
a Group 13 metal precursor, one or more ligands, a solvent, or a
combination thereof to form a precursor mixture; and heating the
precursor mixture to a temperature sufficient to facilitate
formation of the quantum dot.
In any or all of the above embodiments, the Group 11 metal
precursor is a copper-containing precursor, a silver-containing
precursor, or a combination thereof.
In any or all of the above embodiments, the Group 11 metal
precursor is copper acetate, silver acetate, or a combination
thereof.
In any or all of the above embodiments, the Group 13 metal
precursor comprises indium.
In any or all of the above embodiments, the Group 13 metal
precursor is indium acetate.
In any or all of the above embodiments, a metal molar ratio of
Ag:In of 1:2 or a metal molar ratio of Cu:In of 1:2 is used.
In any or all of the above embodiments, the temperature ranges from
150.degree. C. to 200.degree. C.
In any or all of the above embodiments, the temperature is
170.degree. C.
In some embodiments, the methods comprise combining copper acetate,
silver acetate, or a combination thereof with indium acetate, oleic
acid, and dodecanethiol to form a precursor mixture; and heating
the precursor mixture to a temperature ranging from 160.degree. C.
to 180.degree. C. to facilitate formation of a CuInS.sub.2 quantum
dot, an AgInS.sub.2 quantum dot, or a combination thereof.
Embodiments of methods of imaging cells also are disclosed herein,
with such methods comprising: contacting a cell with an imaging
amount of the composite having a core comprising one or more
magnetic nanoparticles, one or more quantum dots, or a combination
thereof and a zwitterionic polymeric coating comprising a
zwitterionic polymer having a structure satisfying a formula
##STR00020## wherein each R.sup.1 independently is selected from
hydrogen or aliphatic; each R.sup.2 is --C(O)Z, wherein Z is
selected from hydroxyl, ether, amine, thiol, or thioether; each
R.sup.3 independently is selected from amide-aliphatic-amine,
amide-aliphatic-amine-aliphatic-carboxylate,
amide-aliphatic-amine-aliphatic-sulfonate, or
amide-aliphatic-thiol; p is an integer selected from zero to 5; q
is an integer selected from zero or 1; and each of a and b
independently is an integer selected from 1 to 200; or a
composition comprising the composite and a biomolecule, a drug, or
both; and detecting cellular update and/or the location of the
composite in the cell.
In any or all of the above embodiments, detecting the location of
the composite comprises exposing the cell to a stain and visually
imaging the cell to detect cellular uptake of the composite.
In any or all of the above embodiments, detecting the location of
the composite comprises analyzing the cell using photoluminescence
spectroscopy to determine the photoluminescent intensity of the
composite.
Also disclosed herein are embodiments of a method of delivering a
therapeutic drug to a subject, comprising: contacting the subject
with a therapeutically effective amount of a composition comprising
the therapeutic drug and a composite having a core comprising one
or more magnetic nanoparticles, one or more quantum dots, or a
combination thereof and a zwitterionic polymeric coating comprising
a zwitterionic polymer having a structure satisfying a formula
##STR00021## wherein each R.sup.1 independently is selected from
hydrogen or aliphatic; each R.sup.2 is --C(O)Z, wherein Z is
selected from hydroxyl, ether, amine, thiol, or thioether; each
R.sup.3 independently is selected from amide-aliphatic-amine,
amide-aliphatic-amine-aliphatic-carboxylate,
amide-aliphatic-amine-aliphatic-sulfonate, or
amide-aliphatic-thiol; p is an integer selected from zero to 5; q
is an integer selected from zero or 1; and each of a and b
independently is an integer selected from 1 to 200.
In any or all of the above embodiments, therapeutic drug is
encapsulated within the zwitterionic polymeric coating.
In any or all of the above embodiments, the drug is doxorubicin,
daunorubicin, epirubicin, idarubicin, or a combination thereof.
VII. Examples
Materials
Poly(maleic anhydride-alt-1-octadecene (PMAO, average M.sub.n
30000-50000), 3-(dimethylamino)-1-propylamine (DMAPA, 99%),
N,N-diisopropylethylamine (DIPEA, 99.5%), .beta.-propiolactone
(Grade II, .gtoreq.90%), gelatin, and poly-D-lysine (PDL) were
purchased from Sigma-Aldrich. 1,3-propanesultone (99%) was
purchased from Alfa Aesar. Tetrahydrofuran (THF, >99%), ethanol
(>99%), methanol (>99%), dichloromethane and chloroform
(>99.9%) were purchased from Pharmco-AAPER. U-87 MG (HTB-14) and
HEK-293 cells (CRL-1537) were ordered from the American Type
Culture Collection (ATCC). RPMI-1640, MEM and DMEM media were from
Corning Cellgro. Paraformaldehyde, Dulbecco's phosphate buffered
saline (DPBS), and phosphate buffered saline (PBS) were from Fisher
Scientific. Heat-inactivated fetal bovine serum (FBS) and stempro
accutase were from Gibco. Bovine serum albumin (BSA) was from MP
Biomedicals. Chlorotoxin (CTX) was purchased from Alomone Labs.
Fluorescein diacetate (FDA), propidium iodide (PI),
4',6-diamidino-2-phenylindole (DAPI) were was from Pierce. All
chemicals or reagents were used as received without further
purification.
The infrared (IR) spectra were acquired using a Perkin-Elmer
Frontier FT-IR spectrometer equipped with Spectrum 10 software and
Universal ATR sampling accessory. The nuclear magnetic resonance
(NMR) spectra were obtained on Varian VNMRS operating at 500 MHz
('H) and 298K. For the zwitterionic magnetofluorescent nanoparticle
preparation, a probe-type Misonix Ultrasonic Liquid Processor
(QSonica) was used. Transmission electron microscope (TEM) images
and Energy-dispersive X-ray (EDX) spectra were acquired using a
JEOL analytical transmission electron microscope (model JEM 2100F)
operated with a 200 kV acceleration voltage and equipped with an
Oxford Energy-Dispersive X-ray (EDX) spectrometer. The optical
characteristics of zwitterionic magnetofluorescent nanoparticles
including ultraviolet-visible (UV-Vis) and photoluminescence
spectra were collected using Shimadzu UV-2450 spectrometer and
Shimadzu RF-5301PC spectrofluorometer. The hydrodynamic sizes were
measured in H.sub.2O using a Malvern Zetasizer Nano ZS dynamic
light scattering (DLS) instrument equipped with a HeNe laser
operating at 632.8 nm and a scattering detector at 173 degrees. For
iron content assay and colloidal stability embodiments, a
Perkin-Elmer microplate reader was used. Cell viability or
cytotoxicity were estimated using a BD Biosciences SORP LSR II flow
cytometer with 4 lasers (405 nm, 488 nm, 561 nm and 640 nm) and 18
fluorescence detectors. Magnetic resonance imaging was performed on
a Bruker BioSpec 7T horizontal bore system. Fluorescent cellular
images were taken using Leica TCS SP8 (DM 6000 CS) confocal
scanning microscope.
Preparation of Zwitterionic Amphiphiles
Generally, the synthesis of zwitterionic amphiphilic polymer
PMAO-carboxybetaine-sulfobetaine involved two steps. In the first
step, PMAO anhydride rings were opened by the primary amine groups
of DMAPA leading to the presence of terminal carboxyl and tertiary
amine groups on the polymer backbone. In the second step, tertiary
amines reacted with .beta.-propiolactone and 1,3-propanesultone to
form carboxybetaine and sulfobetaine groups, respectively. To avoid
possible ring-opening polymerization of .beta.-propiolactone and/or
1,3-propanesultone, the second-step reaction was started in
ice-bath and the total molarity of both .beta.-propiolactone and
1,3-propanesultone was controlled to be slightly higher than that
of tertiary amines (if all anhydride rings in PMAO were opened). Of
note, although the molar ratio between .beta.-propiolactone and
1,3-propanesultone can be tuned, in this embodiment an 1:1 ratio of
them was used to demonstrate the proof of concept on
PMAO-carboxybetaine-sulfobetaine. The synthesis is simple, and its
production yield is >80%.
In one example, PMAO-DMAPA was first prepared by stirring the
mixture of 2.02 g PMAO and 1.1 mL DMAPA in 25 mL CH.sub.2Cl.sub.2
over ice-bath for 3 hours followed by precipitation of the product
by adding acetone. The white precipitate was collected by
filtration and then washed 3 times (with sonication) by dissolving
in .about.15-20 mL CHCl.sub.3 and precipitating with acetone (3
times volume excess). The product was dried under vacuum overnight
resulting in 2.03 g (.about.77% yield) of PMAO-DMAPA. For the
synthesis of PMAO-DMAPA-carboxybetaine-sulfobetaine, the mixture of
0.50 g PMAO-DMAPA, 192 .mu.L DIPEA, 38.4 .mu.L of
.beta.-propiolactone, and 68.2 mg 1,3-propanesultone in 5 mL
CH.sub.2Cl.sub.2 was stirred over ice-bath for .about.3 hours and
then at room temperature overnight. The resulting lightly cloudy
mixture was pipetted into centrifuge tubes and washed (>5 times)
by dissolving in .about.1 mL CHCl.sub.3-MeOH (1:1, v/v),
precipitating with acetone (3-5 times volume) and collecting the
precipitate by centrifugation. The white solid was dried under
vacuum overnight giving 0.54 g of product (.about.88% yield).
The synthesized PMAO derivatives were characterized by FTIR, as
shown in FIG. 2. PMAO-DMAPA spectrum showed the disappearance of
anhydride C.dbd.O at around 1857 cm.sup.-1 and 1776 cm.sup.-1 and
the appearance of new peaks at around 1713 cm.sup.-1 for carboxyl
C.dbd.O, around 1649 cm.sup.-1 for amide C.dbd.O, and around 1560
cm.sup.-1 for amide N--H, which indicates the addition of DMAPA to
PMAO. In the spectrum of PMAO-carboxybetaine-sulfobetaine, the
peaks from PMAO-DMAPA are represented and some new peaks at around
1175 cm.sup.-1 and 1035 cm.sup.-1 are observed. As shown in FIG. 3,
the peaks at around 1175 cm.sup.-1 and 1035 cm.sup.-1 are also
observed in the FTIR spectrum of amidosulfobetaine-16 (ASB-16)
which is an alkyl chain with a sulfobetaine head. These two peaks
represent S.dbd.O in the sulfo group. The spectrum comparison and
analysis indicates the formation of sulfobetaine after the reaction
of the tertiary amines of PMAO-DMAPA with 1,3-propanesultone. Peaks
associated with carboxybetaine in PMAO-carboxybetaine-sulfobetaine
are not significant for observation, probably because the peaks for
the additional carboxyl C.dbd.O from the formed carboxybetaine
groups in PMAO-carboxybetaine-sulfobetaine are overlapping with
those of the existing carboxyl C.dbd.O in PMAO-DMAPA. However, a
slight distortion at around 1590 cm.sup.-1 in the spectrum of
PMAO-carboxybetaine-sulfobetaine (not marked but close to the mark
line at 1560 cm.sup.-1) is still distinguishable. Considering there
is a significant peak at around 1590 cm.sup.-1 for
N-Dodecyl-N,N-(dimethylammonio)butyrate (DDMAB) which is an alkyl
chain with a carboxybetaine head (as shown in FIG. 1), this minor
distortion may suggest the addition of carboxybetaine to PMAO.
The synthesized PMAO derivatives dissolved in a mixture of
CDCl.sub.3 and CD.sub.3OD were further characterized using NMR. All
NMR spectra were presented in FIG. 5. For all samples, the
hydrophobic alkyl chain (C.sub.18) of the polymer was observed at
0.80 ppm for --CH.sub.3 and 1.18 ppm for --CH.sub.2. For
PMAO-DMAPA, two additional peaks are presented at 2.66 ppm and 1.76
ppm. In literature, the following peaks have been reported for the
attachment of DMAPA to polymer backbones: C(O)NHCH.sub.2 at 3.2-3.3
ppm, CH.sub.2N(CH.sub.3).sub.2 at 2.36 ppm, N(CH.sub.3).sub.2) at
2.2 ppm, CH.sub.2CH.sub.2N(CH.sub.3).sub.2 at 1.6 ppm. The observed
peaks due to the addition of DMAPA to PMAO appear in the chemical
shift range as reported in the literature for the same chemical
group. For PMAO-carboxybetaine-sulfobetaine, it has two peaks at
1.88 ppm and 2.75 ppm (similar to these of PMAO-DMAPA), but have an
additional peak at around 3.02 ppm. Literature has reported the
following peaks due to the carboxybetaine addition to polymer
backbones: C(O)NHCH.sub.2 at 2.75-3.48 ppm, C(O)NHCH.sub.2CH.sub.2
at 1.55-2 ppm, CH.sub.2N.sup.+(CH.sub.3).sub.2 at 2.75-3.48 ppm,
N.sup.+(CH.sub.3).sub.2 at 2.9-3.3 ppm, CH.sub.2CH.sub.2COO.sup.-
at 3.4-3.55 ppm, CH.sub.2COO.sup.- at 2.25-2.75 ppm; and the follow
peaks due to the sulfobetaine addition to polymer backbones:
C(O)NHCH.sub.2 at 3.2 ppm, C(O)NHCH.sub.2CH.sub.2 at 1.9 ppm,
CH.sub.2N.sup.+(CH.sub.3).sub.2 at 3.3 ppm, N.sup.+(CH.sub.3).sub.2
at 3.05-3.1 ppm, CH.sub.2CH.sub.2CH.sub.2SO.sub.3.sup.- at 3.4 ppm,
CH.sub.2CH.sub.2SO.sub.3.sup.- at 2.15 ppm, and
CH.sub.2CH.sub.2SO.sub.3.sup.- at 2.89 ppm. The peaks for
PMAO-carboxybetaine-sulfobetaine match with what were reported.
For the synthesis of PMAO-DMAPA-CBSB, PMAO-DMAPA (0.50 g) was first
dissolved in 5 mL of anhydrous CHCl.sub.3 and then cooled over
ice-H.sub.2O bath. Then, DIPEA (192 .mu.L, 1.1 mmole) was added and
the mixture stirred for 5 minutes before adding
.beta.-propiolactone (38.4 .mu.L, 0.55 mmole), and
1,3-propanesultone (67.9 g, 0.55 mmole). The reaction mixture was
stirred over ice-H.sub.2O bath for 3 hours and then at room
temperature overnight. The resulting lightly cloudy mixture was
pipetted into centrifuge tubes and washed (7.times.) by dissolving
in .about.1 mL CHCl.sub.3-MeOH (1:1, v/v), precipitating with
acetone (3-5.times. volume) and collecting the precipitate by
centrifugation. The white solid was dried under vacuum overnight
giving 0.54 g of product.
For the alternative approach for the introduction of carboxybetaine
group, PMAO-CBtBu was first prepared by stirring the mixture of
PMAO-DMAPA (0.50 g), DIPEA (0.40 mL, 2.3 mmol), and tert-butyl
bromoacetate (0.52 mL, 3.4 mmole) in 5 mL of anhydrous CHCl.sub.3
at room temperature overnight. The clear reaction mixture was
poured into 50 mL anhydrous Et.sub.2O to precipitate the product.
The white precipitate was collected and washed with 15 mL Et.sub.2O
(3.times.) and dried under vacuum giving 1.0 g of product. As the
product weighed more than expected, it was re-washed with 10 mL
acetone (3.times.), but much less solid was isolated and the
product was found to have dissolved in acetone, as solids were
collected after rotary evaporation of the acetone filtrate (0.96
g). The product isolated from evaporation of acetone was used
without further purification for the next reaction. It was stirred
with 1.2 mL TFA under Argon for 1 h to hydrolyze the tert-butyl
ester group. The product (PMAO-CB) was precipitated and washed with
anhydrous Et.sub.2O, and then dried under vacuum (0.63 g) and
further washed with acetone giving 0.55 g of white solid after
drying.
In some embodiments, amino intermediates comprising CB and SB units
can be made and then used to open maleic anhydride-containing
polymers. The primary amine of DMAPA was first protected by
reaction with di-tert-butyl dicarbonate (Boc anhydride) to obtain
Boc-DMAPA. The reaction of Boc-DMAPA with .beta.-propiolactone (or
1,3-propanesultone) was followed by acid treatment to remove the
protecting group and obtain the corresponding acid salt of amino-CB
(or amino-SB). The obtained intermediates were characterized by
.sup.1H NMR spectroscopy and confirmed by comparison with
literature data when available.
The obtained Boc-DMAPA was reacted with 1,3-propanesultone, which
was followed by acid hydrolysis of Boc protecting group to get the
corresponding acid salt of amino-SB. The .sup.1H NMR of the product
showed the disappearance of the signal at 1.44 ppm (compared with
Boc-DMAPA) which indicates the removal of the Boc protecting group.
The proton peaks were also assigned as indicated below. The
products/intermediates for the synthesis of amino-SB were also
characterized by FTIR spectroscopy. The addition of
1,3-propanesultone to Boc-DMAPA is indicated by the more prominent
peaks at 1163 cm.sup.-1 (overlapping with C--N vibration) and 1033
cm.sup.-1 for S.dbd.O of the sulfonic group. The removal of the Boc
protecting group is confirmed by the disappearance of the peaks at
1694 cm.sup.-1 and 1528 cm.sup.-1 from the C.dbd.O and N--H,
respectively, of Boc-DMAPA.
The amino-CB was alternatively prepared from the reaction between
Boc-DMAPA and tert-butyl bromoacetate, which is eventually followed
by acid hydrolysis of the tert-butyl groups to give the
corresponding acid salt of amino-CB. The .sup.1H NMR spectrum of
the product showed the disappearance of the tert-butyl groups at
1.54 and 1.45 ppm, indicating the deprotection (removal of Boc and
tert-butyl ester groups) by acid hydrolysis.
The isolated products/intermediates for amino-CB were also
characterized by FTIR spectroscopy. For the reaction of Boc-DMAPA
and t-butyl bromoacetate, the appearance of overlapping peaks at
1740 cm.sup.-1 and 1691 cm.sup.-1 are for the C.dbd.O of butyl
ester and the C.dbd.O of Boc group, respectively. The peaks at 1249
cm.sup.-1 and 1152 cm.sup.-1 are for the C--O vibration of the
t-butyl ester group which disappeared after removal of the
protecting groups via HCl hydrolysis, along with the peak at 1691
cm.sup.-1 from removal of Boc protecting group. The C.dbd.O for
amino-CB (acid salt) is found to have shifted to 1740 cm.sup.-1
corresponding to that for carboxylic acid. The broad peak at 2800
cm.sup.-1, aside from containing the C--H, also represents the O--H
from the carboxylic group.
The protection of the primary amine of DMAPA was done by stirring
(under Ar) the solution of DMAPA (5 mL, 39.3 mmole) in 21 mL
anhydrous MeOH and di-tert-butyl dicarbonate (Boc anhydride, 9.6
mL, 41.4 mmole) over ice/H.sub.2O bath for at least 1 hour and then
allowing mixture to reach room temperature and continued stirring
overnight. Bubbling (from formation of CO.sub.2) occurred during
the addition of Boc anhydride. The resulting clear, colorless
solution was concentrated by rotary evaporation, dissolved in 30 mL
H.sub.2O, and then washed with EtOAc (25 mL.times.5). The combined
EtOAc washes was dried over anhydrous Na.sub.2SO.sub.4, filtered to
remove the drying agent, and the filtrate concentrated by rotary
evaporation. The residue was dried under vacuum giving 5.64 g (71%
yield) of colorless oil.
To synthesize the sulfobetaine intermediate, Boc-DMAPA (2.51 g,
12.4 mmole) was dissolved in 15 mL of anhydrous DMF and then 1.56
mL (2.13 g, 17.4 mmol) of 1,3-propanesultone (1.56 mL, 17.4 mmole)
was added. The clear, colorless solution was stirred at room
temperature for 2 days. The resulting clear reaction mixture was
added to 100 mL of anhydrous Et.sub.2O to precipitate the product,
which came out as viscous material. After cooling the mixture in
the freezer for 1 hour, the lightly cloudy supernatant was
decanted. The wash was repeated (2 more times) by dissolving the
residue in MeCN, adding Et.sub.2O, cooling in the freezer, and
decantation of the supernatant. The viscous residue was transferred
into a pre-weighed flask using MeCN, concentrated by rotary
evaporation, and dried under vacuum overnight giving 4.51 g (112%
yield) of white, foamy residue. The sample was re-washed using MeCN
and Et.sub.2O (3.times.) resulted to 4.39 g (109% yield) of foamy
residue after drying. Although the sample weighed more than
expected, it was used as is for the acid hydrolysis of the Boc
protecting group using aqueous HCl. Boc-DMAPA-SB (4.15 g, 12.8
mmole) was dissolved in 11 mL of H.sub.2O and 5.3 mL of
concentrated HCl (.about.5.times. excess) was added, during which
bubbling occurred. The mixture was stirred (loosely stoppered) at
room temperature overnight. The sample was concentrated by rotary
evaporation (remaining HCl/H.sub.2O was co-evaporated with 10 mL
Et.sub.2O (3.times.)) and the residue dried under vacuum giving
4.21 g of viscous residue. The product was re-stirred with 30 mL of
4 M HCl in MeOH for 4 hours, concentrated with rotary evaporation
(with Et.sub.2O co-evaporation) and then dried under vacuum giving
4.67 g of faintly yellow oil. The sample was used as is for
reaction with PMAO. For the remaining sample, several trials were
done to isolate the HCl salt as a solid. Eventually, the sample
(3.34 g) was dissolved in 10 mL MeOH and CH.sub.2C12/iPrOH/MeOH
(10:5:1, volume ratio) was added in portions (used 45 mL) at which
a solid mass collected at the bottom of the flask. The sample was
washed with CH.sub.2C12/iPrOH/MeOH (10:5:1, volume ratio) and after
drying under vacuum a white solid was obtained. The sample was
transferred into a fitted funnel by repeatedly washing with
CH.sub.2Cl.sub.2/iPrOH/MeOH (10:5:1, volume ratio) (with
sonication), and after drying under vacuum, 2.04 g of white solid
was obtained.
For the synthesis of the carboxybetaine intermediate, Boc-DMAPA
(1.26 g, 6.23 mmole) was dissolved in 6 mL anhydrous MeCN and then
tert-butyl bromoacetate (1.31 mL, 8.72 mmole) was added. The
mixture was stirred at 50.degree. C. (oil bath temperature) for 24
hours. After cooling to room temperature, the reaction mixture was
added to Et.sub.2O to precipitate the product, which came out as
viscous residue. The mixture was cooled in the freezer for 1 hour
and the supernatant was decanted. The wash was repeated (2 more
times) by dissolving the residue in MeCN and adding Et.sub.2O. The
residue was transferred into a pre-weighed flask using MeCN,
concentrated by rotary evaporation, and then dried under vacuum
overnight giving 2.32 g of white, foamy residue. The product was
used as is for the next reaction, which is the acid hydrolysis of
Boc and t-Bu ester groups using aqueous HCl. Boc-DMAPA-CB-tBuester
(2.17 g) was dissolved in 10 mL of H.sub.2O and 4.6 mL of
concentrated HCl (.about.5.times. excess) was added, during which
bubbling occurred. The mixture was stirred (loosely stoppered) at
room temperature for 2 hours. The mixture was concentrated by
rotary evaporation (with Et.sub.2O co-evaporation) and then dried
under vacuum giving 1.58 g of viscous residue. The NMR spectrum
showed that the Boc and t-Bu groups were gone. In the attempt to
recover the product as a solid, the sample was re-stirred with 15
mL of 4M HCl in MeOH for 2 hours, concentrated by rotary
evaporation (with Et.sub.2O co-evaporation), and rinsed with
acetone. After drying under vacuum, solid sample was obtained. The
sample was transferred into a fritted funnel by repeated washing
(with sonication) with Et.sub.2O and then CH.sub.2C12/iPrOH/MeOH
(10:5:1, volume ratio) and then dried under vacuum giving 0.99 g of
white solid.
For the synthesis of PMAO-CBSB, PMAO (100 mg, 2.5 .mu.mole,
.about.285 .mu.mole anhydride) was dissolved in 5 mL anhydrous
CHCl.sub.3. In a separate container, amino-SB (HCl salt, 42 mg) and
amino-CB (HCl salt, 33 mg) were mixed with 500 .mu.L anhydrous MeOH
(some undissolved solid present). DIPEA (300 .mu.L) was added to
convert the intermediates to the free base form, resulting in a
clear, colorless solution. The solution of amino-SB and amino-CB
was added to PMAO and the mixture stirred at room temperature for
24 hours. The reaction mixture was concentrated by rotary
evaporation and the residue dissolved in CHCl.sub.3-MeOH (1:1,
v/v). The product was precipitated by adding acetone and collected
by centrifugation. The dissolution in CHCl.sub.3-MeOH and
precipitation with acetone was repeated 2 more times and the
residue dried under vacuum giving 144 mg of white solid.
Preparation of Zwitterionic Magnetofluorescent Nanoparticles
On the basis of the synthesized PMAO-carboxybetaine-sulfobetaine,
zwitterionic magnetofluorescent nanoparticles integrating small
MnFe.sub.2O.sub.4 magnetic nanoparticles and CuInS.sub.2/ZnS
quantum dots in the micellar cores were fabricated. Magnetic
nanoparticles and quantum dots were around 5 nm. Using the
disclosed methods to produce composites comprising quantum dots,
the quantum dot photoluminescence can be tuned from 650 nm-800 nm
(FIG. 4). In one example, CuInS.sub.2 quantum dots with around 720
nm photoluminescence emission were prepared, and after ZnS shell
growth the CuInS.sub.2/ZnS quantum dots emit photoluminescence at
around 685 nm. A solution of 0.6 mg MnFe.sub.2O.sub.4 magnetic
nanoparticles and 2.4 mg CuInS.sub.2/ZnS quantum dots in THF (900
.mu.L) and 1.7 mg PMAO-carboxybetaine-sulfobetaine in
CHCl.sub.3-MeOH (.about.50 .mu.L) was layered on top of cold water
in a glass vial. The mixture was ultrasonicated using the Misonix
Ultrasonic Liquid Processor with a 5 W output power for 1 minute.
After sonication, the organic solvents were removed by rotary
evaporation at room temperature and the sample filtered through a
0.2 .mu.m syringe filter. Empty micelles or single-nanoparticle
based micelles were removed by centrifugation at 18,000 rpm for 25
min (twice). The collected zwitterionic magnetofluorescent
nanoparticles were dispersed in 400 .mu.L of water, and stored at
4.degree. C. until further use.
The fabricated zwitterionic magnetofluorescent nanoparticles were
characterized using TEM and EDXS. FIGS. 6A and 6B present TEM
images of zwitterionic magnetofluorescent nanoparticles, and the
images indicate the sizes of zwitterionic magnetofluorescent
nanoparticles in around 50.about.60 nm. On the basis of TEM
imaging, the overall size of zwitterionic magnetofluorescent
nanoparticles mostly distributed over a range of 20.about.150 nm
was observed. EDX analysis in FIG. 6C further demonstrates that
zwitterionic magnetofluorescent nanoparticles are composed of Mn,
Fe, O, Cu, In, Zn and S elements. In addition to TEM, DLS data of
zwitterionic magnetofluorescent nanoparticles have been collected
and presented in Table 1. The hydrodynamic sizes of zwitterionic
magnetofluorescent nanoparticles are 99 nm with a standard
deviation at 60 nm. The zwitterionic magnetofluorescent
nanoparticle hydrodynamic sizes mainly are contributed by the
micelle hydrophobic core (imaged by TEM), the polymer shell, and
the hydration layer caused by zwitterions on the polymer shell. The
(Mn+Fe) content in zwitterionic magnetofluorescent nanoparticles
was also quantified on the basis of the iron content determination
of zwitterionic magnetofluorescent nanoparticles using thiocyanate
colorimetry. Table 1 also shows that the (Mn+Fe) recovery rate for
0.6 mg magnetic nanoparticles input to zwitterionic
magnetofluorescent nanoparticles is as high as around 60%.
TABLE-US-00001 TABLE 1 Hydrodynamic sizes and (Mn + Fe) recovery
rates of zwitterionic magnetofluorescent nanoparticles and
zwitterionic magnetic nanoparticles Hydrody- (Mn + Fe) namic sizes
recovery rates Particles (nm) (%) zwitterionic 99 .+-. 60 62 .+-. 1
magnetofluorescent nanoparticles zwitterionic magnetic 133 .+-. 69
60 .+-. 4 nanoparticles
The photoluminescence spectrum of zwitterionic magnetofluorescent
nanoparticles is shown in FIG. 7A, compared to that of quantum dots
in organic solvents. The photoluminescence intensities are scaled
by the quantum yield of zwitterionic magnetofluorescent
nanoparticles relative to that of hydrophobic quantum dots in THF
(the measured quantum yields of zwitterionic magnetofluorescent
nanoparticles are <10%). It can be seen that the
photoluminescence of zwitterionic magnetofluorescent nanoparticles
is significantly quenched. The quenching may be caused by the MNP
absorption on quantum dot emissions, or by the reduction of quantum
dot excitation/emission surfaces due to the blocking effect of
surrounding magnetic nanoparticles. FIGS. 7A and 7B also show that
the overall photoluminescence property of zwitterionic
magnetofluorescent nanoparticles are tunable by adjusting the
MNP:quantum dot mass ratio in the fabrication process. Additional
embodiments are illustrated in FIGS. 8A and 8B. The absorbance of
the prepared zwitterionic magnetofluorescent nanoparticles was
characterized using a UV-Vis spectrophotometer, as shown in FIG. 9.
No significant difference on UV-Vis absorbance curves was observed
for all fabricated zwitterionic magnetofluorescent
nanoparticles.
The magnetic imaging features of magnetofluorescent nanoparticles
were characterized using a magnetic resonance imaging (MRI)
instrument (Bruker BioSpec). The MR images of zwitterionic
magnetofluorescent nanoparticles were acquired with a conventional
spin echo acquisition (TR=6000 ms) with TE values ranging from 9.5
ms to 190 ms. T.sub.2 parameter (or R.sub.2 parameter,
R.sub.2=1/T.sub.2) of zwitterionic magnetofluorescent nanoparticles
was extracted by fitting the exponential decay of the signal
waveform and measuring the signal intensity at a series of
different TE values. FIG. 7B presents R.sub.2 parameter (or
1/T.sub.2) of zwitterionic magnetofluorescent nanoparticles vs
(Mn+Fe) concentration. The relaxivity (r.sub.2) of zwitterionic
magnetofluorescent nanoparticles was calculated as the slope of the
R.sub.2 curve. The relaxivity r.sub.2 value of zwitterionic
magnetofluorescent nanoparticles is around 150 mM.sup.-1s.sup.-1.
R.sub.2 parameter of zwitterionic magnetic nanoparticles (using
magnetic nanoparticles and the synthesized zwitterionic amphiphiles
but not containing any quantum dots) also was measured as a
comparison. The relaxivity r.sub.2 of zwitterionic magnetic
nanoparticles is around 266 mM.sup.-1s.sup.-1. In literature,
T.sub.2 parameter of agglomerated nanomagnet clusters has been
formulated and discussed. Briefly, for agglomerated nanomagnets,
1/T.sub.2=16f.sub.a.DELTA..omega..sup.2.tau..sub.D/45 with f.sub.a
being the volume fraction occupied by the agglomerated nanomagnets,
.DELTA..omega.=.mu..sub.oM.gamma./3 (where .mu..sub.o is the vacuum
magnetic permeability, M is the particle magnetization, and .gamma.
is the proton gyromagnetic ratio), and .tau..sub.D is the
translational diffusion time around the cluster
(.tau..sub.D=R.sub.a.sup.2/D where R.sub.a being the cluster radius
and D being the water diffusion coefficient). Although the formula
of T.sub.2 parameter discussed for the agglomerated system is built
only on small magnetic nanoparticles, it can be applicable to
zwitterionic magnetofluorescent nanoparticles with a mixture of
quantum dots and magnetic nanoparticles. Specifically, f.sub.a
probably can be re-defined as the volumic fraction occupied by
magnetic nanoparticles in zwitterionic magnetofluorescent
nanoparticles. Considering zwitterionic magnetofluorescent
nanoparticles and zwitterionic magnetic nanoparticles have the
similar size ranges (as shown in Table 1), the increase of quantum
dots over magnetic nanoparticles in the fabrication may cause the
decrease of f.sub.a, which further result in the total net
magnetization (M) of zwitterionic magnetofluorescent nanoparticles
to drop. Thus, quantum dots involved in the fabrication cause
R.sub.2 and thus r.sub.2 decrement of zwitterionic
magnetofluorescent nanoparticles. In spite of the R.sub.2 or
r.sub.2 drop, the relaxivity value for zwitterionic
magnetofluorescent nanoparticles is still comparable to many
reported ones. Of note, the fabricated zwitterionic magnetic
nanoparticles also can be considered as excellent contrasts for MRI
because of their high magnetic relaxivity and (Mn+Fe) recovery
rate.
Cell Cytotoxicity of Magnetofluorescent Nanoparticles
In biosensing/imaging applications, aggregation of zwitterionic
magnetofluorescent nanoparticles will cause the degradation or even
loss of their physiochemical and biological functionalities, and
thus zwitterionic magnetofluorescent nanoparticles are expected to
have excellent colloidal stability. The photoluminescence intensity
of zwitterionic magnetofluorescent nanoparticles dispersed in
PBS-5% FBS with pH 4-9, a 1 M NaCl-5% FBS and human serum was
monitored over 72 hours at 37.degree. C. using a microplate reader,
and presented in FIG. 10. With reference to FIG. 10, the different
time periods included in the legend correspond to different bars
present for each pH value shown on the graph, as read from left to
right for the time and for each bar. That is, each group of bars
for each pH value is ordered by increasing time period from left to
right. For example, the left-most bar of each pH bar set
corresponds to 1 hour and the right most bar of each pH bar set
corresponds to 72 hours. The photoluminescence intensity and hence
stability of these zwitterionic magnetofluorescent nanoparticles
was maintained in all these conditions. Moreover, no precipitates
or significant photoluminescence intensity decreases were observed
over one week at 37.degree. C. Stored at 4.degree. C., the
zwitterionic magnetofluorescent nanoparticles were also found to be
stable in water at over at months (FIGS. 11 and 12). As with FIG.
10, each group of bars shown in FIGS. 11 and 12 includes one bar
for each different time period provided by the legend, and time
periods are organized in increasing order so that each bar from
left to right corresponds to an increased time period (e.g., the
left-most bar of each group represents a time period of 1 hour and
the right-most bar of each group represents week 10). The excellent
stability of these zwitterionic magnetofluorescent nanoparticles in
these conditions especially in a solution with pH4, a solution with
1M salinity, and serum, is very attractive. Carboxybetaine groups
and sulfobetaine groups coated on the surface of zwitterionic
magnetofluorescent nanoparticles should be attributed to this
stability. These zwitterions facilitate a hydration layer coating
on zwitterionic magnetofluorescent nanoparticles, and the hydration
layer is very stable and almost remains unperturbed under harsh
conditions such as high/low pH values, high salinity, and complex
matrix. The stability of zwitterionic magnetofluorescent
nanoparticles offers a great deal of flexibility for their
biomedical applications.
The cytotoxicity of the zwitterionic magnetofluorescent
nanoparticles was studied using human primary glioblastoma cells
(U-87 MG) and human embryonic kidney 293 cells (HEK-293). U-87 MG
and HEK-293 represent tumor cells and normal cells, respectively.
FIGS. 13A and 13B show the measured cell viabilities for U-87 MG
and HEK-293 after 24-hour incubation with zwitterionic
magnetofluorescent nanoparticles under different concentrations,
respectively. With reference to FIGS. 13A and 13B, each bar of the
different bar groups represents a different MNP:quantum dot ratio,
with each left-most bar representing an MNP:QD of 1:1, each middle
bar representing an MNP:QD of 1:2, and each right-most bar
representing an MNP:QD of 1:4. An additional embodiment is
illustrated in FIG. 14. With reference to FIG. 14, the left-most
bar of each bar group represents the data for U-87 MG cells, and
the right-most bar of each bar group represents the data for
HEK-293 cells. It can be seen that the cell viabilities under
different particle concentrations are comparable to controls, and
thus the zwitterionic magnetofluorescent nanoparticles are
biocompatible and their cytotoxicity is minimal.
In one example, A U-87 MG human brain glioblastoma cell line was
cultured (37.degree. C., 5% CO.sub.2) on 24-well plastic plates in
MEM medium with 10% FBS overnight. The human embryonic kidney cell
line HEK-293 was cultured (37.degree. C., 5% CO.sub.2) on 24-well
plastic plates in RPMI-1640 medium with 10% FBS overnight. For the
zwitterionic magnetofluorescent nanoparticle cytotoxicity examples,
cells were incubated with zwitterionic magnetofluorescent
nanoparticles in growth medium at various concentrations. After
24-hour incubation, cells were gently rinsed with DPBS and released
from well bottom using stempro accutase, and then stained with FDA
and PI to determine live versus dead cells. Dead cells (red
staining by PI) and live cells (green staining by FDA) were counted
using a BDBiosciences SORP LSR II flow cytometer. The cell
viability was calculated as the ratio of live cells over the sum of
live cells and dead cells.
Conjugation of Composites with Neutravidin for Avidin-Biotin
Binding Assay
To verify that zwitterionic magnetofluorescent nanoparticles are
capable for bioconjugation, zwitterionic magnetofluorescent
nanoparticles with a MNP:quantum dot mass ratio at 1:4 were
fabricated and conjugated with Neutravidin using regular EDC/NHS
cross-linker. The conjugated zwitterionic magnetofluorescent
nanoparticles were then incubated with biotinylated magnetic
microbeads (.about.4 .mu.m in diameter). After incubation,
microbeads were washed and their fluorescence was measured. FIG. 15
shows the fluorescence responses of the microbeads after their
incubation with serial dilutions of Neutravidin-conjugated
zwitterionic magnetofluorescent nanoparticles (FIG. 15, right-most
bars of each bar group) from the stock suspension (or with
different concentrations). As controls, the fluorescence responses
of the biotinylated magnetic microbeads after their incubation with
non-conjugated zwitterionic magnetofluorescent nanoparticles (FIG.
15, left-most bars of each bar group) under the same dilutions were
measured. For each dilution or concentration, the fluorescence
response for non-conjugated zwitterionic magnetofluorescent
nanoparticles is clearly lower than that of conjugated zwitterionic
magnetofluorescent nanoparticles. Thus, zwitterionic
magnetofluorescent nanoparticles can be covalently conjugated with
biomolecules. In addition, the assay result indicates that the
nonspecific binding (NSB) of non-conjugated zwitterionic
magnetofluorescent nanoparticles to microbeads is very low. The low
NSB may be attributed to the nature of zwitterion (for example, the
hydration layer of zwitterions makes zwitterionic
magnetofluorescent nanoparticles more hydrophilic and minimizes the
electrostatic or hydrophobic absorption of zwitterionic
magnetofluorescent nanoparticles on the reaction surface).
In one example, zwitterionic magnetofluorescent nanoparticles with
the MNP:quantum dot mass ratio at 1:4 were prepared and suspended
in 200 .mu.L H.sub.2O. A 50-.mu.L portion of the zwitterionic
magnetofluorescent nanoparticles was mixed with 7.2 .mu.g EDC (37.5
nmole in PBS pH 7.4), 8.1 .mu.g sulfo-NHS (37.5 nmole in PBS pH
7.4), 13.6 .mu.L Neutravidin (1.13 nmole in H.sub.2O), and the
total volume brought to 100 .mu.L with PBS pH 7.4. The mixture was
incubated for 2 hours at room temperature. After incubation, the
magnetofluorescent nanoparticles were washed by centrifugation
using PBS pH 7.4 (3.times.). The residue was dispersed in 200 .mu.L
PBS pH 7.4.
Three .mu.L of biotinylated magnetic microbeads (4.5 .mu.m
diameter, 4.times.108 beads/mL) were placed in each well of a
microplate. Different volumes of the conjugate stock solution were
loaded into wells, and PBS pH 7.4 buffer was used to bring the
total volume in each well to 50 .mu.L. The unconjugated
zwitterionic quantum dot stock solution under the same dilutions
was used as controls. The microplate was vortexed at room
temperature for 1 hour, and then the magnetic microbeads in each
well were washed using PBS pH 7.4 with 1% BSA and dispersed in 50
.mu.L of PBS pH 7.4. The fluorescence signals of the suspended
microbeads in wells were measured using a microplate reader at the
excitation wavelength of 405 nm and the emission wavelength of 655
nm. The examples were performed in triplicates.
Tumor Cell Targeting Using Peptide Conjugated Zwitterionic
Magnetofluorescent Nanoparticles
For cellular imaging studies, CTX was used as a targeting ligand.
CTX is a 36-amino acid peptide that specifically binds to matrix
metalloproteinase II (MMP-2) present on the surface of glioma cells
with high affinity. The specific binding results in loss of
gelatinase activity, disruption in chloride channel currents,
reduction in both MMP-2 and chloride channel expressions, and
internalization of chloride channels. U-87 is a human primary
glioblastoma cell line expressing MMP-2 receptors, and CTX can
specifically bind to and be internalized into U-87. To investigate
whether CTX-conjugated zwitterionic magnetofluorescent
nanoparticles can be specifically targeted to and internalized into
U-87, a nonmalignant cell line human embryonic kidney 293 (HEK-293)
was used as a control. FIGS. 16A-16D present the representative
overlaid confocal images demonstrating the cellular
uptake/internalization when each type of cells were incubated with
CTX- and non-conjugated zwitterionic magnetofluorescent
nanoparticles under the same concentration of particles (i.e., 0.1
.mu.g (Mn+Fe)/mL). Corresponding to each overlaid image in FIGS.
16A-16D, FIG. 17 shows the associated confocal images at different
channels. FIG. 16E shows fluorescence intensity per unit cytoplasm
area (counting>200 cells) under a series of zwitterionic
magnetofluorescent nanoparticle concentrations. It can be seen that
U-87 cells do internalize more CTX-conjugated zwitterionic
magnetofluorescent nanoparticles than HEK-293, and non-conjugated
zwitterionic magnetofluorescent nanoparticles produce no
significant cellular uptake by both cell lines. Through this
comparison, it can be concluded that CTX-conjugated zwitterionic
magnetofluorescent nanoparticles are specific to U-87. It was also
observed that HEK-293 did internalize some CTX-conjugated
zwitterionic magnetofluorescent nanoparticles at high
concentrations. It is believed that the cellular uptake of
CTX-conjugated micelles may involve pinocytosis mechanisms in high
concentration ranges.
In one example, zwitterionic magnetofluorescent nanoparticles were
conjugated with CTX via EDC/sulfo-NHS mediated reaction. Briefly,
60 .mu.L of the collected zwitterionic magnetofluorescent
nanoparticles were reacted with 50 .mu.g CTX with the assistance of
EDC/sulfo-NHS in PBS for 2.about.3 hours. The CTX-conjugated
zwitterionic magnetofluorescent nanoparticles were washed using
centrifuge, suspended in 250 .mu.L PBS, and stored at 4.degree. C.
before use. A U-87 MG human brain glioblastoma cell line was
cultured (37.degree. C., 5% CO.sub.2) on glass coverslip coated
with gelatin in MEM medium with 10% FBS until 50-80% confluency was
achieved. The human embryonic kidney cell line HEK-293 was cultured
(37.degree. C., 5% CO.sub.2) on glass coverslip coated with PDL
(poly-D-lysine) in RPMI-1640 medium with 10% FBS until 50.about.80%
confluency was achieved. For the zwitterionic magnetofluorescent
nanoparticle tumor cell targeting examples, cells were incubated
with CTX conjugated zwitterionic magnetofluorescent nanoparticle in
DMEM with 2% BSA at various concentrations. As control, cells were
also incubated with non-conjugated zwitterionic magnetofluorescent
nanoparticles. After 2 hours of incubation, cells were gently
rinsed three times with PBS, fixed with 4% PFA in PBS solution for
20 minutes and washed three times with PBS. For cellular nuclei
staining, cells were incubated with DAPI, washed three times with
PBS, and then mounted on glass slides. Cells were imaged using a
Leica confocal microscope and images were analyzed using ImageJ.
The statistical significance (p<0.05) was determined by the
single-tailed student t test.
Methods of Making Quantum Dots Using Thermal Decomposition
Silver Acetate (99%), Indium (III) Acetate (99.99%), Zinc Stearate
(ZnO: 12.5-14%), and Paraformaldehyde (97%) were purchased from
Alfa Aesar. Sulfur (>99.99%), Trioctylphosphine (TOP, 90%),
1-Dodecanethiol (98%), 1-Octadecene (ODE, 90%), Oleic Acid (99%),
Methanol (99.93%), and 1,10-Phenanthroline (99%) were purchased
from Sigma Aldrich. Tetrahydrofuran (THF, >99%), Ethanol
(>99%), Chloroform (>99.9%), and Hexane (95%) were purchased
from Pharmco-AAPER. Methoxy poly(ethylene
glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) (MW.about.5000:5000
Da) and maleimide-PEG-PLGA (MW.about.5000:5000 Da) were purchased
from Akina, Inc. Dulbecco's Phosphate Buffered Saline (DPBS),
Phosphate Buffered Saline (PBS), Ethylenediaminetetraacetic Acid
(EDTA), Acetonitrile (99.96%) and Traut's Reagent were from Fisher
Scientific. Heat-inactivated Fetal Bovine Serum (FBS) was from
Gibco. U-87 MG and HEK-293 cells were ordered from the American
Type Culture Collection (ATCC). RPMI-1640, MEM and DEMEM media were
from Corning Cellgro. 7-Aminoactinomycin D dye (7-AAD, excitation
at 488 nm and emission at 647 nm) for cell nucleic acid staining
was from Invitrogen. Chlorotoxin (CTX) was purchased from Alomone
Labs. Bovine Serum Albumin (BSA) was from MP Biomedicals. Zeba spin
desalting columns (MWCO 7k) were from Pierces.
The ultraviolet and visible (UV-Vis) spectra of materials were
obtained with a UV-Vis spectrometer (UV-2450 from Shimadzu).
Photoluminescence spectra of quantum dots in organic-phase and
aqueous-phase were acquired using a spectrophotometer (RF-5301PC
from Shimadzu) Photoluminescence intensity of quantum dots in
various buffers was obtained using a microplate reader (PerkinElmer
2030 equipped with a 535 nm emission filter and a 405 nm excitation
filter). Transmission electron microscope (TEM) images and
Energy-dispersive X-ray (EDX) spectra were acquired using a JEOL
analytical transmission electron microscope (model JEM 2100F
operated with a 200 kV acceleration voltage) equipped with an
Oxford Energy-Dispersive X-ray (EDX) spectrometer. X-ray
Diffraction (XRD) data was collected by a coupled Theta:2-Theta
scan on a Rigaku Ultima-III diffractometer equipped with Copper
X-ray tube with Ni beta filter, parafocusing optics,
computer-controlled slits, and D/Tex Ultra 1D strip detector. The
hydrodynamic sizes of micelles were measured using a dynamic light
scattering (DLS) instrument (Malvern Zetasizer Nano ZS) equipped
with a HeNe laser operating at 632.8 nm and a scattering detector
at 173 degrees. Probe sonication was performed with a Misonix
ultrasonic processor (QSonica S-4000) equipped with a microtip.
Infrared (IR) spectra of materials were collected using a Fourier
transform infrared (FT-IR) spectrometer (Perkin-Elmer Frontier)
with Spectrum 10 software and the Universal ATR Sampling Accessory.
Cells were imaged using a Leica confocal microscope and images were
analyzed using ImageJ.
Quantum yields of quantum dots were calculated according to the
following equation, using standard references including Rhodamine
6G (emission peak at 556 nm, QY=95% in ethanol) and Oxazine 170
(emission peak at 640 nm, QY=63% in methanol),
QY.sub.S=QY.sub.R.times.(I.sub.S/I.sub.R).times.(A.sub.R/A.sub.S).times.(-
n.sub.S/n.sub.R).sup.2
where QY.sub.S and QY.sub.R are the quantum yields of sample and a
standard reference, respectively; I.sub.S and I.sub.R are the
integrations of fluorescence emissions of sample and a standard
reference, respectively; A.sub.S and A.sub.R are the corresponding
absorbance of sample and a standard reference, respectively; and
n.sub.S and n.sub.R are the refractive indices of the corresponding
solvents.
During quantum yield measurements, the absorbance of each sample or
each standard reference deviated by less than 0.1. For each sample,
the standard reference with the most similar absorption and/or
luminescence characteristics was chosen for quantum yield
measurements.
AIS/ZnS Quantum Dot Synthesis
For a typical synthetic reaction, silver acetate (0.1 mmol), Indium
(III) acetate (0.2 mmol), DDT (4 mL) and oleic acid (0.2 mmol, 63.5
.mu.L) were added in a three-necked round bottom flask equipped
with a condenser and magnetic stir bar. This mixture was degassed
under vacuum for 20 minutes at 130.degree. C. until the solution
became clear. The solution temperature was then increased to
170.degree. C. under a flow of Argon. As the temperature was
increased, the color of the reaction solution changed gradually
from yellow to orange, indicating the nucleation and growth of AIS
quantum dots. Small amounts of the reaction solution (0.1-0.2 mL)
were collected using a syringe at different time intervals and
injected into hexane in clean vials to terminate growth of quantum
dots. All solutions collected from the studies were diluted in a
quartz cuvette with hexane for UV-Vis absorbance and
photoluminescence measurements. After the reaction was complete,
the solution was cooled to room temperature. The crude quantum dots
solution was purified repeatedly with the solvent combinations of
hexane/ethanol and chloroform/acetone by centrifugation and dried
under vacuum.
For ZnS shell growth, the Zn precursor was prepared by mixing zinc
stearate (1.6 mmol) and ODE (4 mL) in a round-bottom flask. The
mixture was gradually heated to .about.100.degree. C. with stirring
under vacuum until no vigorous bubbling was observed. The
temperature was increased to 160.degree. C. under argon until a
clear solution was obtained. The sulfur precursor was prepared by
dissolving sulfur (1.6 mmol) in DDT (3.2 mL) and TOP (0.8 mL). The
ZnS shell coating of AIS quantum dots was carried out in situ
without purification of the core. 4 mL ODE was added to the crude
AIS solution. This core solution was degassed under vacuum at
130.degree. C. for 30 min and then to 210.degree. C. under Argon.
Both zinc and sulfur precursors were injected in sequence 5 times
to the core growth solution at 210.degree. C. in 0.5 mL portions at
15 min intervals. After reactions were complete, mixtures were
cooled down to room temperature and AIS/ZnS quantum dots were
purified using hexane/ethanol and chloroform/acetone, and dried
under vacuum. It is possible to collect around 40 mg AIS quantum
dots and around 100 mg AIS/ZnS quantum dots per reaction.
FIG. 18 shows photoluminescence spectra of AIS quantum dots during
the time course of growth at 170.degree. C. It can be seen that the
quantum yield of AIS quantum dots is enhanced during growth
reaching 13% at 75 min. The quantum yield enhancement over time is
likely caused by a reduction of quantum dot core defects during
heat treatment. Nevertheless, the photoluminescence spectral shift
is not significant or sensitive to growth time. This optical
characteristic suggests that AIS growth is very slow. The slow
growth of AIS quantum dots is probably due to the relatively mild
reaction temperature. When using a higher reaction temperature
(230.degree. C.), typically for CIS quantum dots, it was found that
the products of the AIS synthetic system are hard to solubilize in
chloroform and hexane, and thus hard to characterize optically.
Characterization of Quantum Dots
AIS quantum dots and AIS/ZnS quantum dots were further
characterized using TEM. FIGS. 19A and 19B present TEM and high
resolution TEM (HRTEM) images of AIS and AIS/ZnS quantum dots,
respectively. TEM images of CIS quantum dots made using thermal
decomposition method embodiments disclosed herein are shown in FIG.
34A (normal resolution) and 34B (high resolution). The HRTEM images
reveal crystalline patterns and sizes (.about.4.5 nm for AIS and
.about.6 nm for AIS/ZnS) of both quantum dots, indicating that the
synthesized particles to be nanocrystals. Energy-dispersive X-ray
(EDX) spectra confirm that AIS/ZnS quantum dots are composed of Ag,
In, Zn, and S (FIG. 19C) and AIS quantum dots are composed of Ag,
In and S (FIG. 20). More specifically, Table 1 shows the elemental
atomic ratios of AIS and AIS/ZnS quantum dots. For AIS quantum
dots, the atomic ratio between Ag and In is close to 1:1 with the
atomic percentage of In slightly higher. For AIS/ZnS quantum dots,
the atomic percentages of Ag and In drop off but the Ag percentage
is reduced to a greater extent, and the atomic percentages of Zn
and S are increased. The significant reduction of Ag atomic
percentage in AIS/ZnS is probably due to Zn etching to replace Ag
during the ZnS shell growth. It is generally agreed that cation
exchange between Zn ions (from Zn precursor) with Ag (or Cu) in AIS
(or CIS) causes photoluminescence blue shift and quantum yield
enhancement. As shown in FIG. 1, both effects were observed after
growing ZnS shell on AIS cores. EDX analysis demonstrates a good
match of the observed photoluminescence blue shift and quantum dot
brightness enhancement of AIS/ZnS quantum dots compared to AIS
quantum dots.
The crystal phase of the AIS and AIS/ZnS quantum dots was examined
by X-ray powder diffraction (XRD), as shown in FIG. 21. The XRD
pattern of AIS quantum dots shows three broad peaks at
2.theta.=26.8.degree., 44.6.degree. and 52.2.degree., which can be
assigned respectively to the diffractions of the (112), (204) and
(312) planes of the tetragonal AgInS.sub.2. No other phases or
impurities were observed. The AIS/ZnS quantum dots diffraction
pattern shows a similar profile to that of the AIS quantum dots
with three right-shifted peaks at 2.theta. of 27.7.degree.,
46.0.degree. and 54.0.degree.. These peaks can be seen at positions
intermediate between the diffractions of (112), (204), and (312)
planes of tetragonal AgInS.sub.2 and the diffractions of (111),
(220), and (311) planes of cubic ZnS, suggesting that Zn atoms were
deposited on or diffused into the surface of the AIS cores.
It should be noted that all characterized materials MS and AIS/ZnS
quantum dots were synthesized with an original Ag:In precursor
molar ratio of 1:2. This ratio can be used to avoid possible side
products or black precipitates in reactions. AIS quantum dots were
synthesized at different Ag:In precursor molar ratios (Ag:In=1:1,
1:2, and 1:4) without changing other conditions. The collected AIS
samples were diluted in hexane and their photoluminescence and
UV-Vis absorbance spectra were measured. As shown in FIG. 22, all
samples have similar photoluminescence spectra without obvious
shifts. However, as illustrated in FIG. 23, AIS quantum dots
synthesized with Ag:In=1:1 have a wider absorbance wavelength range
than the other two samples. Moreover, the AIS quantum dot solution
(Ag:In=1:1) is a dark brown color, as illustrated in the inset of
FIG. 23. This suggests that there is something else (possibly
Ag.sub.2S) produced during the reaction with Ag:In=1:1 at
170.degree. C. but particles are not large enough to precipitate.
The AIS quantum dot solution (Ag:In=1:1) was further used to grow
the ZnS shell on AIS quantum dots and as shown in FIG. 24A, the
resultant product includes both a green solution and black
precipitates. However, after the ZnS shell growth of AIS quantum
dots (Ag:In=1:2), the resultants are clear (FIG. 24B). These
observations lead to the molar ratio of Ag:In=1:2 used in the
synthesis. AIS synthesis using Ag:In=1:4 was not performed, because
this ratio does not significantly shift quantum dot
photoluminescence and excess In precursors in the reaction increase
synthesis cost.
In the proposed thermal decomposition approach, indium acetate and
silver acetate could initially react with DDT to form intermediate
compounds of Ag(SC.sub.12H.sub.25).sub.x and
In(SC.sub.12H.sub.25).sub.x upon heating and are dissolved in DDT.
As the reaction temperature is raised to 170.degree. C., the
intermediate compounds could act as precursors and further are
decomposed into Ag--S and In--S to form Ag--In--S particles.
Considering that there are some unexpected materials produced in
the reaction with a molar ratio of Ag:In at 1:1, it is possible
that Ag(SC.sub.12H.sub.25).sub.x compounds are decomposed faster
than In(SC.sub.12H.sub.25).sub.x compounds and thus Ag--S are
excess to form dark Ag.sub.2S particles. This could explain why 1:2
molar ratio of Ag:In is good for the reaction. Of note, the
photostability of the produced AIS and AIS/ZnS quantum dots were
tested and presented in FIGS. 25A and 25B. It can be seen that
AIS/ZnS quantum dots remain their photostability but AIS quantum
dots are significantly photobleached. The photobleaching of AIS
quantum dots could be caused by complex UV-induced chemical
reactions at the interface between AIS and organic solvents. For
example, electrons excited from AIS react with organic solvents to
form free radicals which could further etch the AIS surface and
quench the AIS photoluminescence. The photostability of AIS/ZnS
quantum dots is believed to benefit from the protection of AIS core
by its ZnS shell.
To demonstrate potential biomedical applications of the produced
AIS/ZnS quantum dots, these quantum dots were loaded into the core
of PLGA-PEG based micelles to form quantum dot-micelles. The
quantum dot-micelle preparation is schematically illustrated in
FIG. 26. Briefly, AIS/ZnS quantum dots were mixed with amphiphilic
polymers of PLGA-PEG in organic solvents, and then dispersed them
into water with sonication. During the vacuuming or
water-replacement of organic solvents, the hydrophobic portion
(PLGA) of the polymers and quantum dots are self-associated into a
semi-solid core, and the hydrophilic portion (PEG) of the polymers
forms a coronal layer. Photoluminescence spectra of AIS/ZnS quantum
dots before and after water transfer via micelles were measured and
are shown in FIG. 27. It can be seen that the quantum dot-micelles
show a slight red-shift in photoluminescence spectra and remain
around 30% quantum yield compared to quantum dots suspended in
organic solvents. The red-shift and the quantum yield drop after
the phase transfer is believed to be caused by a compact quantum
dot cluster in micelle cores. In the compact cluster, the emission
photons from smaller quantum dots are absorbed by larger quantum
dots to emit at longer wavelengths (a Forster resonance energy
transfer process). Moreover, the cluster reduces the total
excitation and emission surface area of quantum dots, resulting in
a quantum yield drop. In spite of quenching, the quantum
dot-micelles are still adequate for optical imaging applications.
The insets of FIG. 27 show the hydrodynamic sizes (126 nm.+-.53 nm)
of the quantum dot-loaded micelles measured by DLS and the TEM
image of quantum dot-micelles, respectively. FIGS. 28A and 28B show
the TEM image and high resolution TEM image of an individual
micelle. It can be seen that some dots are presented in a single
micelle, and with further zooming into the micelle crystal lattices
of dots are observed. Further EDS analysis on micelles (FIG. 28C)
shows that silver, indium, zinc and sulfur elements are presented
in micelles, which are the composites of AIS/ZnS quantum dots.
Preparation of AIS/ZnS Micelles
The solution of 2.4 mg AIS/ZnS quantum dots and 9.6 mg PEG-PLGA
(50% PEG-PLGA and 50% maleimide-PEG-PLGA) in THF/acetonitrile was
layered on the top of cold water in a glass vial. The mixture was
ultrasonicated using the Misonix Ultrasonic Liquid Processor with a
3 W output power for 1 minute. After sonication, THF/acetonitrile
was removed by rotary evaporation at room temperature and the
sample filtered through a 0.2 .mu.m syringe filter to remove large
aggregates. Empty micelles or single-nanoparticle based micelles
were removed by centrifugation at 18,000 rpm for 15 minutes. The
collected micelles were then re-filtered through a 0.2 .mu.m
syringe filter, concentrated using a centrifugal filter, dispersed
in 400 .mu.L of water, and stored at 4.degree. C. until further
use.
Cell Culture, Cellular Uptake/Internalization of AIS/ZnS Micelles,
and Cellular Imaging
A U-87 MG human brain glioblastoma cell line (ATCC HTB-14) was
maintained in Minimum Essential Media (MEM, Corning Cellgro)
supplemented with 10% FBS at 37.degree. C. and 5% CO.sub.2. The
human embryonic kidney cell line HEK-293 (ATCC CRL-1537) was
maintained in RPMI-1640 medium (Corning Cellgro) supplemented with
10% FBS at 37.degree. C. and 5% CO.sub.2.
For the quantum dot-micelle cellular uptake assay, U-87 and HEK-293
cells were plated on 24-well plastic plates and allowed to
propagate for 2-3 days until they reached 50-80% confluency. The
cells were incubated with CTX-conjugated or non-conjugated AIS/ZnS
quantum dot-micelles in cell culture medium with 2% BSA for 2 hours
at 37.degree. C. After incubation, the solution was removed and the
cells were washed three times with cold PBS buffer (pH 7.4). Then
cells were fixed with 4% paraformaldehyde in PBS at room
temperature for 20 minutes, followed by cell nucleus staining with
7-Aminoactinomycin D (7-AAD) for 45 minutes. The cells were
examined by a laser scanning confocal microscope (LSCM, Leica, TCS
SP8, Germany). The statistical significance (p<0.05) was
determined by the single-tailed student t test.
To demonstrate potential biomedical or biological applications of
AIS/ZnS quantum dots, these quantum dots were loaded into the core
of PLGA-PEG (5 kDa:5 kDa) based micelles to form the AIS/ZnS
quantum dot-micelles and further investigated their
specific-targeting functionality as cellular imaging probes or
contrasts. With functional groups such as maleimide on PEG heads,
the quantum dot-micelles were conjugated with chlorotoxin (CTX), a
ligand that specifically binds to U-87 brain tumor cells. Cellular
imaging studies showed that the quantum dot-micelles conjugated
with CTX are specifically internalized into the brain tumor cells.
Since the hydrophobic core of micelles can be loaded with both
drugs and image contrasts, the specific cellular internalization
suggests that the quantum dot-micelle structures could be important
and versatile nanoplatforms for cell- or tissue-based diagnosis and
therapy. Moreover, loading multiple quantum dots in the hydrophobic
cores could avoid the blinking effect of single quantum dot and
therefore facilitate continuous image tracking. Notably, the common
surface modification approaches using thiolated ligands to exchange
hydrophobic ligands capped on quantum dots (CdSe or CdS) are less
favorable for AIS/ZnS quantum dots. This is because quantum dots,
including AIS/ZnS quantum dots, are usually capped with the strong
coordinating ligand dodecanethiol which is hard to displace by
other foreign thiols. Although some groups have reported the use of
amphiphilic polymers to encapsulate quantum dots (CdSe or CdS),
they need an overnight air-dry process followed by a heated film
hydration, or a 24-hour dialysis to remove organic solvents or
excess amphiphilic polymers. The heated hydration or the prolonged
dialysis may be incompatible with certain functional groups (e.g.,
maleimide) on polymers for bioconjugation. Moreover, these studies
used cadmium-based quantum dots, which have been concerns for
biomedical applications and environments. Loading cadmium-free
AIS/ZnS quantum dots into PLGA-PEG based micelles for biomedical
applications is simple and fast and effectively avoids these
potential limitations.
For CTX conjugation, the thiolation of CTX was completed by
dispersing 12.5 nmol of CTX in 100 .mu.L of PBS (pH8, 5 mM EDTA)
with 125 nmol of Traut's reagent in 9 .mu.L of PBS (pH 8, 5 mM
EDTA) for 1 hour at room temperature. A volume of 87 .mu.L of the
prepared micelles in PBS (pH 6.7) was incubated with the thiolated
CTX for 2-3 hours at room temperature. The resultant CTX-AIS/ZnS
micelle product was purified using Zeba spin desalting columns
(MWCO 7k) equilibrated with PBS, resuspended in 400 .mu.L of PBS as
a stock solution and stored at 4.degree. C. before use. AIS/ZnS
micelles without CTX conjugation were used as controls.
For cellular imaging studies, CTX was used as a target ligand and
used U-87 MG brain tumor cells as model cells. CTX is a 36-amino
acid peptide that was originally isolated from scorpion venom, and
specifically binds to tumors of neuroectodermal origin. Further
studies have demonstrated that CTX is a specific matrix
metalloproteinase II (MMP2) inhibitor and can bind with MMP2
present on the surface of glioma cells with high affinity. The
specific binding results in loss of gelatinase activity, disruption
in chloride channel currents, reduction in both MMP2 and chloride
channel expressions, and internalization of chloride channels.
Recent studies have also implicated annexin A2 (ANXA2) as a new
recognition target of CTX in multiple tumor cell lines, which may
activate similar uptake mechanisms as those of MMP2. U-87 is a
human primary glioblastoma cell line expressing MMP2 receptors, and
CTX can specifically bind to and be internalized into U-87. In this
example, quantum dot-micelles were conjugated with CTX via a
maleimide-thiol reaction. The CTX conjugation was confirmed by
Fourier transform infrared spectroscopy, as shown in FIG. 29. The
spectrum of the AIS/ZnS quantum dot-micelles exhibits the
characteristics of both the alkyl chains in oleic acid and
dodecanethiol from AIS/ZnS quantum dots, corresponding to the
CH.sub.2 stretching vibrations peaks at 2922 cm.sup.-1 and 2852
cm.sup.-1, and the ester carbonyl in PLGA from PLGA-PEG,
corresponding to the C.dbd.O peaks at 1757 cm.sup.-1. After CTX
conjugation with the quantum dot-micelles, a new peak at 1650
cm.sup.-1 corresponding to the N--H band of primary amines in
lysine and arginine residues of CTX, appears in the spectrum of
CTX-conjugated quantum dot-micelles. This appearance indicates the
successful conjugation of CTX with the quantum dot-micelles.
In order to verify that (i) CTX conjugated quantum dot-micelles can
be internalized into U-87 cells and (ii) the internalization is due
to the CTX-MMP2 interaction, two sets of examples were performed.
In the first, U-87 cells were incubated with CTX conjugated quantum
dot-micelles spiked in DEME with 2% BSA for 2 hours. In the second,
400 .mu.M 1,10-phenanthroline was added to DEME, keeping all other
conditions same. 1,10-phenanthroline is a broad-spectrum MMP2
inhibitor and can disrupt the MMP2 activity by chelating and
removing Zn ions from the catalytic domain of MMP2. As a result,
1,10-phenanthroline can block the interaction between CTX and MMP2
and thus the sequential cellular internalization process.
After incubation, the U-87 cells from two sets of examples were
washed, fixed, stained and imaged. Representative confocal images
shown in FIG. 30A demonstrates the cellular uptake/internalization
of CTX-conjugated quantum dot-micelles by U-87 cells. The
representative confocal images in FIG. 30B illustrate the quenching
effect of 1,10-phenanthroline on uptake/internalization. FIG. 30C
shows quantitative data (the fluorescence intensity from the
internalized quantum dot-micelles per unit cytoplasm area
counting>200 cells) comparing cellular uptake/internalization
and the quenching effect under the different concentrations or
dilutions of CTX-conjugated quantum dot-micelles (100-800 times
dilution). These results demonstrate that the internalization of
CTX-conjugated quantum dot-micelles into U-87 cells is caused by an
interaction between CTX and MMP2.
To further confirm that CTX-conjugated quantum dot-micelles is
specific to U-87, human embryonic kidney 293 cells (HEK-293), a
nonmalignant cell line that does not express MMP2 and ANXA2 on cell
surfaces, was used as controls to examine their response to quantum
dot-micelles conjugated with/without CTX and compared to those of
U-87. FIGS. 31A and 31B and FIGS. 31C and 31D show the
representative cellular uptake images (overlaid confocal images)
for U-87 and HEK-293, respectively, under the same quantum
dot-micelle concentration or dilution. FIG. 31E shows fluorescence
intensity (from the internalized quantum dot-micelles) per unit
cytoplasm area under a series of dilutions of quantum dot-micelles
stock solutions. It can be seen that U-87 cells do internalize more
CTX-conjugated quantum dot-micelles (FIG. 31E, left-most bar of
each bar group) than HEK-293 (FIG. 31E, third bar from the left for
each bar group), and non-conjugated quantum dot-micelles (FIG. 31E,
second and fourth bars from the left for each bar group) produce no
significant cellular uptake by both cell lines. Through this
comparison, it can be concluded that CTX-conjugated quantum
dot-micelles are specific to U-87. Interestingly, it was also
observed that HEK-293 did internalize some CTX-conjugated quantum
dot-micelles at a high concentration. Some pioneer work has
reported that CTX can increase the rate of pinocytic
internalization. The cellular uptake of CTX-conjugated micelles may
involve pinocytosis mechanisms in high concentration ranges.
Using PLGA-PEG to wrap AIS/ZnS quantum dots is an effective
approach for phase transfer and bio-applications. The AIS/ZnS
quantum dot-micelles can be used as image contrasts or probes can
be used to detect endogenous targets expressed on brain tumor
cells, or more broadly to cell- or tissue-based diagnosis and
therapy. Similar quantum dot-micelles also can be constructed using
the zwitterionic polymeric coatings described herein.
Synthesis of Cu:AIS Quantum Dots
Ag(DDTC) (0.1 mmol), In(Ac).sub.3 (0.2 mmol) and DDT (4 mL) were
added in a three-necked round bottom flask equipped with a
condenser and magnetic stir bar. This mixture was degassed under
vacuum for 30 min at 125.degree. C. until the solution became
clear. The solution temperature was then increased to
190.about.200.degree. C. under a flow of argon and held at this
temperature for 10 min to grow AIS cores. After the reaction was
completed, the solution was cooled to room temperature, and 4 mL of
ODE was added to the AIS core growth solution without any
purification. Then the solution was heated to 120.degree. C. under
vacuum for 20 min and then to 190.about.200.degree. C. under argon.
Copper precursors prepared by dissolving CuI (0.2 mmol) in DDT (8
mL) were injected into the solution for the formation of Cu:AIS
quantum dots. After 10 min growth, the solution was cooled to room
temperature. The nanocrystal solution was purified repeatedly with
the solvent combinations of hexane/ethanol and chloroform/acetone
by centrifugation and then dried under vacuum. For the synthesis of
Cu:AIS quantum dots with different Cu doping concentrations, the Cu
initial concentrations ([Cu]/([Ag]+[In])) were changed in the range
from 0.about.10 mol %. Small amounts of the reaction solution
(0.1.about.0.2 mL) were collected using a syringe at different time
intervals and injected into hexane in clean vials to terminate
growth of quantum dots. All solutions collected from the example
were diluted in a quartz cuvette with hexane for UV-Vis absorbance
and photoluminescence measurements.
Synthesis of Cu:AIS/ZnS Quantum Dots
For ZnS shell growth, the zinc precursor was prepared by dissolving
zinc stearate (0.4 mmol) in ODE (4 mL) at 140.degree. C., and the
sulfur precursor was prepared by dissolving sulfur (0.4 mmol) in
DDT (3.2 mL) and TOP (0.8 mL). The growth of the ZnS shell on
Cu:AIS quantum dots was conducted in situ without purification of
the core solution. Once the growth of cores was completed, the
nanocrystal solution was heated up to 210.degree. C. under argon.
Then both zinc and sulfur precursors were injected in sequence 3
times to the Cu:AIS growth solution in 0.5 mL portions at 15 min
intervals. After the reaction was finished, mixtures were cooled
down to room temperature and Cu:AIS/ZnS quantum dots were purified
using hexane/ethanol and chloroform/acetone, and dried under
vacuum.
Quantum yields of quantum dots were calculated according to the
following equation, using standard references including Rhodamine
6G (emission peak at 556 nm, QY=95% in ethanol) and Oxazine 170
(emission peak at 640 nm, QY=63% in methanol),
QY.sub.S=QY.sub.R.times.(I.sub.S/I.sub.R).times.(A.sub.R/A.sub.S).times.(-
n.sub.S/n.sub.R).sup.2 where QY.sub.S and QY.sub.R are the quantum
yields of sample and a standard reference, respectively; I.sub.S
and I.sub.R are the integrations of fluorescence emissions of
sample and a standard reference, respectively; A.sub.S and A.sub.R
are the corresponding absorbance of sample and a standard
reference, respectively; and n.sub.S and n.sub.R are the refractive
indices of the corresponding solvents. In quantum yield
measurements, the absorbance of each sample or each standard
reference deviated by less than 0.1. For each sample, the standard
reference with the most similar absorption and/or luminescence
characteristics was chosen for quantum yield measurements.
Amphiphile-Encapsulated Cu:AIS/ZnS Composite Probes for Cellular
Imaging
6.67% Cu:AIS/ZnS quantum dots were encapsulated using zwitterionic
polymeric coatings using methods disclosed above. 2.4 mg
Cu:AgInS.sub.2/ZnS quantum dots in THF (900 .mu.L) and 1.6 mg
zwitterionic amphiphiles in CHCl.sub.3-MeOH (64 .mu.L) were
dispersed into water under sonication. After sonication, the
organic solvents were removed by rotary evaporation at room
temperature and the sample was filtered through a 0.2 .mu.m syringe
filter. Empty micelles or single-nanoparticle based micelles were
removed by centrifugation at 18 000 rpm for 25 min. The collected
micelles were dispersed in 400 .mu.L of water as the micelle
stock.
60 .mu.L of the micelle stock was reacted with 0.3 mg RGD in borate
buffer for 2.about.3 hours after the activation of carboxyl groups
on micelles' surface using EDC/Sulfo-NHS. The RGD-conjugated
micelles were washed by centrifugation, suspended in 200 .mu.L
borate, and stored at 4.degree. C. before use as the conjugate
stock. The same reaction was done for the conjugation of micelles
with RAD peptide, and the RAD-conjugated micelles were used as a
negative control. Non-conjugated micelles were also used as a
control. A U-87 MG human brain glioblastoma cell line was cultured
(37.degree. C., 5% CO.sub.2) on glass coverslips coated with
gelatin in MEM medium with 10% FBS until 50.about.80% confluency
was achieved. The human embryonic kidney cell line HEK-293 was
cultured (37.degree. C., 5% CO.sub.2) on glass coverslips coated
with PDL (poly-D-lysine) in RPMI-1640 medium with 10% FBS until
50.about.80% confluency was achieved. For the specific cell
targeting examples, cells were incubated with RGD-conjugated
micelles in DMEM with 2% BSA at various concentrations or
dilutions. As controls, cells were also incubated with
RAD-conjugated micelles and non-conjugated micelles. After 3 h
incubation, cells were gently rinsed three times with PBS, fixed
with 4% PFA in PBS solution for 20 minutes and washed three times
with PBS. For cellular nuclei staining, cells were incubated with
DAPI, washed three times with PBS, and then mounted on glass
slides. Cells were imaged using a confocal microscope. Two-way
ANOVA was applied to detect the differences among two cell lines.
SAS (Statistical Analysis Software) 9.4 was used for data
analysis.
Cu atoms were doped into AIS composites through a surface doping
strategy--AIS quantum dots were first grown via thermal
decomposition and then Cu precursors were injected into the AIS NC
solution to react with AIS quantum dots. FIG. 35A presents the
effect of Cu concentrations on the photoluminescence spectra of Cu
doped AIS quantum dots (Cu:AIS quantum dots). It can be seen that
the photoluminescence peak wavelength shows a continuous red shift
from around 600 to 660 nm as Cu concentration in reaction is
increased from 0 to 6.67% (molar percentage), and the red shift is
not significant for Cu concentration above 6.67% or at 10%. The
corresponding UV-vis absorption spectra of these Cu:AIS quantum
dots are shown in FIG. 35B As the Cu concentration increases, the
absorbance edges of these samples are monotonously shifted to
longer wavelength. The absorption edge of 10% Cu:AIS is similar to
that of 6.67% Cu:AIS. This observation is consistent with their
photoluminescence spectra. In FIG. 35A it can also be seen that
quantum yields of Cu:AIS quantum dots decrease from around 30% to
20% with the increase of Cu concentrations from 0% to 6.67%.
Quantum yield of 10% Cu:AIS quantum dots even decrease further even
though the photoluminescence is not red shifted. Through further
comparing the photoluminescence spectra of Cu:AIS quantum dots, it
can be seen that their shapes are also affected by Cu
concentrations. The shape dependence on Cu concentration implicates
that Cu atoms are incorporated into NC lattices and cause some
changes of the lattice energy band or contribute to NC
photoluminescence mechanisms. Of note, as shown in the inset of
FIG. 35A the photoluminescence of non-doped or pure AIS quantum
dots prepared in the thermal decomposition is hard to be tuned by
the NC growth time. It can be seen that Cu doping is an effective
way to tune nanocrystal photoluminescence.
The reason to adopt surface doping is that the optical property of
quantum dots is more controllable than that using homogenous
doping. With 3.33% Cu:AIS quantum dots as a demonstration, FIG. 36A
shows the evolution of their photoluminescence spectra in the time
course of reaction through surface doping. It can be seen that
around 45 nm red-shift occurs at 5 min after Cu injection. With
further prolonged reaction time, no remarkable change in
photoluminescence is observed with respect to peak wavelength and
quantum yield. The similar observation is also noticed in their
absorption spectra as shown in FIG. 36B This demonstrates that the
Cu doping of AIS quantum dots is a fast and reliable process.
However, with the same starting materials, if Cu precursor is mixed
with silver and indium precursors in DDT for thermal decomposition,
the photoluminescence of the produced quantum dots is observed to
red shift to around 650 nm and then blue shift to around 600 nm, as
shown in the inset of FIG. 36A. Moreover, the photoluminescence
spectra become broader in the time course of reaction. It is
possible that the final products could have both sub-populations of
doped AIS quantum dots and non-doped (or intrinsic) AIS quantum
dots. Clearly, surface doping is a better approach to control the
NC optical property.
To further understand the optical property of Cu:AIS quantum dots,
these quantum dots were first characterized using XRD, TEM and EDX.
Three representative samples (pure AIS, 1.67% Cu:AIS and 6.67%
Cu:AIS) were investigated. As shown in FIG. 37A, the XRD pattern of
the pure AIS quantum dots can be indexed with a tetragonal
AgInS.sub.2 crystal. Three apparent diffraction peaks observed at
20=26.8, 44.7, and 52.6.degree. can be assigned to diffractions
from the (112), (204) and (312) planes of the tetragonal phase.
Compared to the pure AIS quantum dots, diffraction patterns of
Cu:AIS quantum dots slightly shifts to the higher angle side and
approaches the 20 values of the tetragonal CuInS.sub.2 (CIS) along
with the increase of Cu concentration. Since the ionic radius of Cu
(0.74 nm) is smaller than that of Ag (1.14 nm), the observed shifts
of the XRD patterns indicate the gradual replacement of Ag with Cu
to form Cu:AIS quantum dots instead of forming individual CIS
quantum dots. These three samples were further characterized using
TEM. FIG. 37B-37D present their TEM and high-resolution TEM (HRTEM)
images. TEM images of AIS and Cu:AIS quantum dots show that their
sizes are at 4.about.5 nm, indicating that the incorporation of Cu
does not significantly influence the particle size. The HRTEM
images show lattice plane spacings of 0.342, 0.340, and 0.332 nm
for AIS, 1.67% Cu:AIS, and 6.67% Cu:AIS quantum dots, respectively.
The values of these spacings are corresponding to (112) planes
determined from diffraction peaks at around 26-28.degree. of XRD
patterns in FIG. 37A. Both HRTEM and XRD data reveal that the
obtained Cu:AIS quantum dots are not a mixture of individual AIS
and CIS phases, but a Cu:AIS crystal. The energy dispersive X-ray
(EDX) spectra further confirms that AIS quantum dots are composed
of Ag, In, and S and Cu:AIS quantum dots are composed of Cu, Ag, In
and S. The resultant elemental compositions of AIS and Cu:AIS
quantum dots are summarized in Table 1. For pure AIS quantum dots,
the atomic ratio between Ag and In is close to 1:1. For Cu:AIS
quantum dots, as Cu concentration is increased in the reaction,
more Cu atoms are doped into quantum dots but the atomic percentage
of Ag decreases. This observation indicates that Cu atoms enter the
AIS structure and partially replace Ag atoms. The reason for the
preferential substitution of Ag instead of In probably is that the
Ag--S bond is weaker than the In--S bond. More Cu atoms doped into
quantum dots could cause more structure or surface defects
quenching the NC photoluminescence for lower quantum yields. The
EDX analysis of 10% Cu:AIS quantum dots was also conducted and the
result shows that the element atomic ratio of Cu:Ag:In:S is
5.1:16.4:27.3:51.2. Compared to the element atomic ratio for 6.67%
Cu:AIS quantum dots (as shown Table 1), more Cu atoms are observed
in 10% Cu:AIS quantum dots. It is possible that the Cu level in AIS
quantum dots may get saturated for 6.67% Cu:AIS quantum dots, and
thus with a higher Cu concentration in doping, additional Cu atoms
may be absorbed on NC surface to quench photoluminescence but not
affect the position of photoluminescence peak.
To enhance the photoluminescence quantum yield, AIS and Cu:AIS
quantum dots were passivated with a shell of ZnS to form AIS/ZnS
and Cu:AIS/ZnS quantum dots. The quantum yields of AIS/ZnS, 1.67%
Cu:AIS/ZnS and 6.67% Cu:AIS quantum dots are around 52.6%, 52.6%,
56.5% quantum yields, respectively. All of them present 30.about.50
nm blue shift in their photoluminescence spectra after ZnS coating.
FIG. 38A shows digital photographs of Cu:AIS and Cu:AIS/ZnS quantum
dots suspended in organic solvents under a UV lamp. It can be seen
that the emission colors of cores and core/shell structures are
Cu-concentration dependent. FIG. 38B demonstrates the typical
photoluminescence and absorption spectra of 6.67% Cu:AIS quantum
dots before and after ZnS coating. FIG. 38C shows that the XRD
diffraction pattern of the 6.67% Cu:AIS/ZnS quantum dots has a
similar profile to that of 6.67% Cu:AIS cores, and shows a medium
phase of Cu:AIS and ZnS crystals. The three main peaks shift to the
high angle side and locate in the middle position of the pattern of
Cu:AIS quantum dots and the standard pattern of cubic ZnS, which
further suggests that Zn atoms are deposited or diffused to the
surface of the Cu:AIS cores. FIG. 38D shows the corresponding TEM
and HRTEM images. The HRTEM image also reveals that the quantum
dots have a clear lattice fringe with a plane spacing of 0.310 nm.
The lattice planet spacing is close to that of the (111) plane in
cubic ZnS quantum dots. Similar to the core samples, Cu:AIS/ZnS
quantum dots have a size around 4.about.5 nm. Probably, the ZnS
coating is mainly dominated by zinc etching into core quantum dots.
As confirmed by the EDX spectrum of 6.67% Cu:AIS/ZnS quantum dots,
the elemental composition ratios of Cu, Ag, In, Zn, and S obtained
from EDX analysis were 2.1%, 9.5%, 19.8%, 12.7%, and 55.8%,
respectively. Both the atomic percentages of Ag and In decrease,
however, the amount of Ag is reduced to a greater extent. At the
same time, the atomic percentage of Zn is increased. The more
reduction of Ag atomic percentage in Cu:AIS/ZnS quantum dots is
probably due to Zn etching to preferentially replace Ag during the
ZnS shell growth. The Cu concentration in the core/shell structures
was observed to be is slightly lower than that of the Cu:AIS cores.
Such results suggest that in the Cu doping process, a portion of Cu
atoms may sit on the surface of AIS cores and be replaced by zinc
atoms during ZnS coating or zinc etching, and another portion of Cu
atoms may diffuse into an inner layer of AIS lattice which cannot
be replaced by zinc but still affect the photoluminescence of
core/shell quantum dots.
FIG. 39A shows the decay curves for AIS, 1.67% Cu:AIS, and 6.67%
Cu:AIS quantum dots. FIG. 39B presents the decay curves for their
corresponding core/shell structures. For each decay curve, a
biexponential function (l(t)=A1e.sup.-t/.tau.1+A2e.sup.-t/.tau.2)
was used to fit the curve. .tau.1 and .tau.2 are the short and long
lifetime parameters, respectively. A1 and A2 are the amplitudes of
the decay components at t=0. Table 2 lists the extracted
characteristics parameters (.tau.1, .tau.2, A1, and A2) for all
investigated samples. According to literature, the
photoluminescence lifetime parameters of AIS quantum dots could be
associated with different electron-hole recombination pathways or
mechanisms. After light excitation, electrons will be relaxed from
conduction bands to surface trap states and donor states. The short
lifetime is be attributed to electron transition from surface trap
states (caused by surface defects) to valence bands. The long
lifetime is attributed to electron transition from donor states to
acceptor states, which results in the broad emission peaks of AIS
quantum dots. From Table 2, it is observed that upon comparing A1
parameter for each core and its corresponding core/shell structure,
A1 parameter is decreased and .tau.1 is increased after ZnS shell
growth. Considering the ZnS shell growth on each core causes
quantum yield enhancement, it is reasonable to attribute the
decrease of A1 to the minimization of surface defects. The
prolonged .tau.1 of core/shell structures probably is associated
with electron transition from near-surface trap states or deep trap
states to valence bands. The near-surface trap states or deep trap
states could be closer to the donor states in energy levels and
thus have a relatively longer lifetime for electrons. Upon
examining the effect of Cu concentrations on A1 and .tau.1 of AIS
cores, it can be seen that A1 of Cu:AIS cores is decreased compared
to that of non-doped AIS cores. According to the literature model,
the A1 decrease should implicate the minimization of surface
defects and thus the enhancement of quantum yield upon Cu doping.
However, FIG. 35A show that Cu doping causes the decrease of
quantum yields. Moreover, as shown in FIG. 35A with low Cu
concentrations, a shoulder in the photoluminescence spectrum is
presented at the right side of the main photoluminescence peak. As
Cu concentration increases, the shoulder is shifted to the left
side of the main photoluminescence peak. As shown in Table 2, Cu
doping also prolongs the average photoluminescence lifetime of
quantum dots. It seems that although the literature model can well
explain the effect of ZnS coating as well as some phenomena of
non-doped or intrinsic AIS quantum dots, a more sophisticated model
or a different viewpoint is needed to explain the photoluminescence
mechanisms of Cu:AIS quantum dots as well as the reason for the
prolonged average photoluminescence lifetime upon Cu doping.
According to literature, the decrease of quantum yields with the
increase of Cu doping levels is probably due to two reasons: (i)
more defects in Cu:AIS lattice are created; (ii) high doping
concentration causes closer distance between Cu atoms and cause
strong Cu--Cu interaction and photoluminescence quenching. The
transition/change of the photoluminescence spectrum shape versus Cu
concentration in doping may be caused by multiple electron-hole
recombination paths. According to literature, Cu doping could
introduce additional Cu T.sub.2 states in the bandgap of AIS
quantum dots. Meanwhile, our material characterization shows that
Cu can etch and replace Ag in AIS host quantum dots, and the doped
Cu could form a Cu--In--S(CIS) layer on AIS surface. The formed CIS
layer could possess some nature of CIS bandgap, and new
donor-acceptor pairs from CIS structures could be existing. The new
CIS donor-acceptor pairs also are energy levels incorporated in the
bandgap of AIS hosts. On the other hand, AIS also has its own
donor-acceptor pairs. As a result, there could be several
electron-hole recombination paths. The photoluminescence of quantum
dots should be a synergistic effect of all these recombination
paths. Cu T.sub.2 states and/or new CIS donor-acceptor pairs are
Cu-concentration dependent, and thus gradually they can be more
dominant than AIS donor-acceptor pairs as the Cu concentration
increases in the doping process. As a result, the change of the
photoluminescence spectrum shape versus Cu concentration can be
observed. With respect to the prolonged average photoluminescence
lifetime, due to the incorporation of Cu T.sub.2 states, the
excited-state lifetime of the dopant emission is longer than the
excitonic emission and the surface emission. Thus, Cu doped quantum
dots gain a prolonged photoluminescence lifetime. The new CIS
donor-acceptor pairs also are energy levels in the AIS bandgap and
they could function as Cu T.sub.2 states to prolong the average
photoluminescence lifetime. Of note, these T.sub.2 states and/or
new CIS donor-acceptor pairs should be the causes for the emission
at longer wavelengths (or red-shift in the doping process). Through
this measurement on photoluminescence decay, it is also good to
know that the average photoluminescence lifetime of the produced
quantum dots is in the range of 300.about.500 ns. The quantum dots
with long lifetimes are attractive for bioimaging--as bioimaging
probes, they can be scanned using microscopy not only in regular
intensity-based fluorescence mode but also in time-resolved
fluorescence mode. Specifically, in the time-resolved mode, the
long lifetime can distinguish NC fluorescence signals from the fast
decaying autofluorescence in cells/tissue, and ensure quantum dots
for more sensitive imaging.
TABLE-US-00002 TABLE 2 Nanocrystals .tau..sub.1/ns A.sub.1
.tau..sub.2/ns A.sub.2 .tau..sub.avg/n- s AIS 49 51.6% 369 48.4%
329 1.67% Cu:AIS 62 45.2% 413 54.8% 374 6.67% Cu:AIS 66 44.8% 414
55.2% 374 AIS/ZnS 125 49.2% 416 50.8% 350 1.67% Cu:AIS/ZnS 131
41.7% 461 58.3% 405 6.67% Cu:AIS/ZnS 151 42.2% 499 57.8% 436
To demonstrate potential biomedical applications of Cu:AIS/ZnS
quantum dots (i.e., specific targeting to brain tumor cells),
composites with 6.67% Cu:AIS/ZnS quantum dots and a zwitterionic
polymer coating were formed through self-assembling. Human primary
glioblastoma U-87 MG cells and human embryonic kidney 293 cells
(HEK-293) are cell lines used in this example. The cytotoxicity of
the micelles was first studied using these two cell lines. Cells
were incubated with micelles in growth medium with 10% FBS at
various concentrations for 48 hours at 37.degree. C. After
incubation, the growth medium was removed and the cells released
from well bottom using stempro accutase, and then stained with
FDA/PI to determine live vs dead cells using flow cytometry (Dead
cells are red staining by PI and live cells are green staining by
FDA). The cell viability was calculated as the ratio of live cells
over the sum of live cells and dead cells. FIG. 40 shows the
measured cell viabilities for U-87 MG (right-most bar for each bar
group) and HEK-293 (left-most bar for each bar group) after 48-hour
incubation with micelles under different concentrations. It can be
seen that for each cell line, the cell viabilities under all
different micelle concentrations are around 95%, which are
comparable to that of controls (no micelles in incubation). Thus
the micelles loaded with Cu:AIS quantum dots are biocompatible. On
the basis of the cytotoxicity example, these micelles were further
conjugated with RGD and RAD peptides via EDC/Sulfo-NHS mediated
reaction. RGD can specifically target to integrin .alpha.v.beta.3
overexpressed on U-87 MG cells and thus it is specific to U-87 MG
cells. RAD with a molecular structure similar to RGD but is
nonspecific to U-87 MG cells and thus used as a control. HEK-293
not expressing integrin .alpha.v.beta.3 is used as a cell line
control. In cellular uptake studies, each cell line was incubated
with the RGD-conjugated micelles, RAD-conjugated micelles, and
non-conjugated micelles under difference concentrations or
dilutions in MEM medium with 2% BSA for 3 hours at 37.degree. C.
After incubation, cells were gently rinsed three times with PBS,
fixed with 4% PFA in PBS solution for 20 minutes and washed three
times with PBS. Afterwards, cells were incubated with DAPI for
cellular nuclei staining, washed three times with PBS, and then
mounted on glass slides. The mounted cells were then imaged using a
Leica confocal microscope and images were analyzed using ImageJ.
FIGS. 41A-41F shows representative cellular uptake images (overlaid
confocal images) for U-87 and HEK-293. Quantitative data
counting>100 cells for each experimental condition were
presented in FIG. 42. It can be seen that for each micelle
concentration or dilution, U-87 cells internalize more
RGD-conjugated micelles (FIG. 42, third bar from the left for each
bar group) than HEK-293 (FIG. 42, sixth bar from the left for each
bar group). RAD-conjugated (FIG. 42, second and fifth bars from the
left for each bar group) and non-conjugated micelles (FIG. 42,
left-most and fourth bars from the left for each bar group) had no
significant cellular uptake by any of cell lines. Clearly, the
micelles using Cu:AIS/ZnS quantum dots can be applied to the
detection of endogenous targets expressed on brain tumor cells. In
this embodiment, the micelles were scanned in regular
intensity-based fluorescence mode. Scanning also could be conducted
in time-resolved fluorescence mode to achieve better image quality
with respect to signal/noise ratio. In addition, radioactive Cu may
be doped into AIS quantum dots so that the achieved quantum dots
have dual imaging functionalities for positron emission tomography
(PET) and luminescence imaging. Considering the hydrophobic core of
micelles can also be loaded with both drugs and image contrasts,
the specific cellular internalization suggests that the
cadmium-free Cu:AIS/ZnS-micelles may also load drugs to be
versatile nanoplatforms for cell- or tissue-based diagnosis and
therapy.
Effect of Chloride Surface Passivation on AgInS.sub.2 (AIS) QDs
In these examples, AIS quantum dots re synthesized using
In(Ac).sub.3 and then are mixed with InCl.sub.3 solution at
130.degree. C. (InCl.sub.3 dissolved in a mixture of oleic acid and
1-octadecene). The controls used as comparisons can be synthesized
using In(Ac).sub.3 and then mixing with In(Cl).sub.3 solution at
130.degree. C. ((In(Ac).sub.3 dissolved in a mixture of oleic acid
and 1-octadecene). At 130.degree. C., such a temperature will not
trigger any reactions but promote the diffusion of chloride to
quantum dot surfaces. In some examples, AIS QDs were synthesized
using silver nitrate (0.1 mmol), In(Ac).sub.3 (0.2 mmol), 1-DDT (8
mL) and oleic acid (250 .mu.L) at 175.degree. C. Then, the quantum
dot solution was cooled down from 175.degree. C. and maintained at
130.degree. C. under an Ar flow. 0.1 mmol InCl.sub.3 or
In(Ac).sub.3 was dissolved in a mixture of oleic acid and
1-octadecene at 130.degree. C. 2.5 mL of InCl.sub.3 or In(Ac).sub.3
solution was injected into the quantum dot solution for 60 min
stirring. The quantum dot solutions were sampled for quantum yield
measurements before and after the injection of InCl.sub.3 or
In(Ac).sub.3 solution. The data are illustrated graphically in
FIGS. 44A and 44B, wherein FIG. 44A shows that with chloride
treatment, the quantum dots reach around 40% enhancement, and FIG.
44B shows that without chloride treatment, the quantum yield of the
quantum dots remains unchanged.
Synthesis of Mn-Doped AIZS/ZnS Composites
Ag(DDTC) (0.1 mmol), In(Ac)3 (0.2 mmol) and DDT (4 mL) were added
into a 50 mL three-necked round bottom flask equipped with a
condenser and a magnetic stir bar. The mixture was heated to
125.degree. C. under vacuum until a clear solution was obtained,
and to 200.degree. C. under a flow of argon for 10 minute reaction.
Then, 0.5 mL of Zn precursors (0.4 mmol zinc stearate in 4 mL of
ODE) was added drop-wise into the AIS core solution to form AIZS
nanocrystals. Afterwards, 0.25, 0.75 or 1.25 mL of Mn precursors,
which were prepared by dissolving 0.1 mmol Mn(Ac).sub.2 in the
mixture of 0.75 mL of ODE and 0.25 mL of OAm, were added drop-wise
into the AIZS nanocrystal solution and the solution was maintained
at 240.degree. C. for 15 minutes. Subsequently, for the ZnS shell
coating or nanocrystal surface etching by zinc, 2 mL of Zn
precursors were slowly added drop-wise to the Mn-doped AIZS growth
solution (cooled to 200.degree. C.) and the solution was then
maintained at 200.degree. C. After the reaction was complete, the
mixture was cooled down to room temperature. The Mn:AIZS/ZnS
nanocrystals were purified repeatedly with the solvent combinations
of hexane/ethanol and chloroform/acetone by centrifugation and then
dried under vacuum. Undoped AIZS/ZnS nanocrystals were prepared in
the way as described above but without any addition of Mn
precursors. Results obtained from this example are shown in FIGS.
45A-45C, 46, 47A-47D, and 48.
Thermal stability study was performed on these Mn-doped ternary
nanocrystals. A small amount of 0.075 mmol-Mn-doped AIZS/ZnS
nanocrystals were dissolved in ODE and DDT (DDT is a native ligand
coated on nanocrystals during synthesis, and it is thus here used
as surfactant for nanocrystals). After vacuum and nitrogen filling,
the solution was heated from room temperature (RT) to 170.degree.
C. and then back to room temperature. In this example, temperatures
higher than 170.degree. C. were avoided to avoid thermal
decomposition of DDT. As shown in FIG. 49, after 50.degree. C., the
photoluminescence of the solution apparently fades with a slight
blue-shift (towards yellow emission) in the time course of the
temperature rising to 170.degree. C. The photoluminescence
(intensity and wavelength) of the solution recovers as the
temperature is back to RT. This is a reversible photoluminescence
quenching. Other two types of Mn:AIZS/ZnS nanocrystals were also
tested and presented the similar behavior to that of 0.075
mmol-Mn:AIZS/ZnS nanocrystals. The results are illustrated in FIG.
49.
In another example, Mn-doped AZIS nanocrystals (schematically
illustrated in FIG. 50A) were prepared using 0.2 mmol Zn precursor,
0.2 mmol In precursor, 0.025 mmol Mn precursor, 0.8 mmol sulfur
precursor, and Ag precursor in the range of 0.025.about.0.1 mmol.
After Mn atoms were doped into AZIS nanocrystals, a Zn precursor
and sulfur precursor were added into the reaction flask to grow a
ZnS shell on nanocrystals to form Mn-doped AZIS/ZnS nanocrystals.
As references, AZIS/ZnS nanocrystals using the exact same
conditions above but without any Mn precursors in synthesis were
made. Results from analyzing nanocrystals formed in this example
are shown in FIGS. 50B and 50C.
In this example, a composite comprising I(II)-III-VI nanocrystals
was made having a composite structure similar to that illustrated
schematically in FIG. 51A (though a person of ordinary skill in the
art with the benefit of this disclosure recognizes that the
synthesized composite may have more or fewer zwitterionic ligands
that the four illustrated in FIG. 51A). The solution of 0.6 mg
MnFe.sub.2O.sub.4 magnetic nanoparticles and 2.4 mg I(II)-III-VI
nanocrystals in THF (900 .mu.L) and 1.7 mg PMAO-CBSB in
CHCl.sub.3-MeOH (.about.50 .mu.L) was layered on top of cold water
in a glass vial. The mixture was ultrasonicated using the Misonix
Ultrasonic Liquid Processor with a 5 W output power for 1 minute.
After sonication, the organic solvents were removed by rotary
evaporation at room temperature and the sample filtered through a
0.2 .mu.m syringe filter. Empty micelles or single-nanoparticle
based micelles were removed by centrifugation at 18,000 rpm for 25
min (twice). The collected ZW-MFNP conjugates were dispersed in 400
.mu.L of water, and stored at 4.degree. C. until further use.
Representative magnetic resonance image and optical images obtained
from such composites at difference concentrations are provided by
FIG. 51B. In another example, composites were further coupled with
chlorotoxin. As shown by FIG. 52, such conjugated composites are
biocompatible with different cell lines (e.g., U87, HEK293, and
activated THP1).
In another example, U87 cells were co-incubated with
chlorotoxin-conjugated conjugates (7.2 mg NC/mL) and 1 mM
LysoTracker (ex465/em535, a fluorescent marker from Invitrogen for
secondary endosomes or lysosomes) for 3 hours and 24 hours,
respectively. Afterward, U87 cells were fixed and stained with
DPAI, and then scanned using confocal microscopy. Representative
overlay images for 3 and 24 hr incubation times are shown in FIGS.
53A and 53B, respectively. Spot provided by these images represent
micelles (smaller peripheral spots relative to the large spots),
endosomes/lysosomes (smaller peripheral spots relative to the large
spots) and nuclei (large spots). Also indicated in FIGS. 53A and
53B is the co-localization of micelles with endosomes/lysosomes.
Such co-localization is qualitative, and it is affected by the
intensity the different peripheral spots in these images. Mander's
and Costes's methods, which are implemented in ImageJ as Plugin
Coloc2, were used for colocalization analysis. These approaches are
not affected by the intensity difference of two colors or probes,
and automatically eliminate image backgrounds caused from
autofluorescence, nonspecific labeling of dyes or markers, and
probe fluorescence arising from out-of-focus image planes. With
ImageJ, the colocalization ratio defined as (pixels of
red).sub.colocalized/(pixels of red).sub.total is found to be
(0.78.+-.0.25) for 3 hours incubation and (0.10.+-.0.07) for 24
hours incubation, counting 10.about.20 cells to analyze the
colocalization ratio (in some studies 10.about.20 cells were
counted). This ratio indicates that the endosome/lysosome escape
efficiency, which is defined as (1-colocalization ratio), is low in
the early stage of micelle uptake by cells, but high with longer
incubation times.
Table 3 lists the element atomic ratio for each investigated
nanocrystals using EDX. With the increase of Mn levels in doping,
the mole fractions of Mn atoms (Mn/(Ag+In+Zn+Mn+S)) that are
incorporated in nanocrystals are increased--0.52%, 2.46%, and 2.96%
for 0.025 mmol-, 0.075 mmol-, and 0.125 mmol-Mn-doped nanocrystals,
respectively.
TABLE-US-00003 TABLE 3 Measured Atomic Ratio by EDX Nanocrystals Mn
Zn Ag In S AIZS/ZnS -- 15.16 11.92 16.56 56.36 0.025
mmol-Mn:AIZS/ZnS 0.52 21.48 10.71 18.70 48.60 0.075 mmol-Mn:
AIZS/ZnS 2.46 16.40 8.32 18.13 54.69 0.125 mmol-Mn:AIZS/ZnS 2.96
19.56 12.99 13.46 51.05
As shown in Table 4, the fast and slow decays for 0.075
mmol-Mn:AIZS/ZnS nanocrystals are corresponding to .tau..sub.1 at
around 40 .mu.s and .tau..sub.2 at around 614 .mu.s. These
lifetimes are much longer than those of undoped AIZS/ZnS
nanocrystals measured at their own photoluminescence peak
wavelength. Moreover, the photoluminescence decays of 0.075
mmol-Mn:AIZS/ZnS nanocrystals measured at different wavelengths
(550, 585, 700 nm) also present their own .tau..sub.1 and
.tau..sub.2 in tens of microseconds and hundreds of microseconds.
Without being limited to a single theory, it currently is believed
that a thin layer of Mn was formed in host AIZS/ZnS nanocrystals,
and that Mn clusters or concentrated Mn dopants may exist in this
layer. Experimentally, photoluminescence quenching with Mn doping
in nanocrystals was observed, which indicates Mn-related defect
formation. The fast decay of 0.075 mmol-Mn:AIZS/ZnS nanocrystals
could result from radiative decay from trap states (caused by Mn
related lattice strains or defects) to the valence band. The trap
states could be aligned or overlapping with Mn 3d states, so that
excited electrons in conduction band could be relaxed to the trap
states directly or through Mn 3d states as relay states. The fast
decay of the nanocrystals also could be from the emission of Mn
dopants but with Mn--Mn spin coupling due to concentrated Mn in the
Mn-doped layer. Additionally, because the AIZS/ZnS nanocrystals act
as host nanocrystals, the fast decay may also arise from alloying
of Mn with AIZS/ZnS nanocrystals in a thin layer. That means, the
new alloys may have new bandgap structures or electron-hole
recombination paths. It is also possible that the photoluminescence
of 0.075 mmol-Mn:AIZS/ZnS nanocrystals is a synergistic effect of
various photoluminescence mechanisms. Notably, from Table 4, the
intensity percentage of the slow decayed photoluminescence (i.e.,
A2) is low as several percent over the whole photoluminescence
intensity.
Other nanocrystals with different Mn doping were measured in a
similar way as 0.075 mmol-Mn:AIZS/ZnS nanocrystals. Their
photoluminescence lifetime parameters are also listed in Table 4.
The decay behavior of 0.125 mmol-Mn:AIZS/ZnS nanocrystals is very
similar to that of 0.075 mmol-Mn:AIZS/ZnS nanocrystals. 0.025
mmol-Mn:AIZS/ZnS nanocrystals have smaller .tau..sub.1 and A2
parameters compared to the other two types of nanocrystals, and
thus they present short average photoluminescence lifetimes.
According to Table 3, the Mn level in 0.025 mmol-Mn:AIZS/ZnS
nanocrystals is much less than that in other two types of
nanocrystals. The small .tau..sub.1 and A2 for 0.025
mmol-Mn:AIZS/ZnS nanocrystals could be related to the low Mn level
in the hosts. Interestingly, according to the literature, a lower
Mn level in host nanocrystals should produce a longer
photoluminescence lifetime probably because the Mn--Mn distance is
long. Such a conflict indicates that the Mn emission with Mn--Mn
coupling could be existing but not be the major photoluminescence
mechanism for the fast decay (corresponding to .tau..sub.1) in
Mn:AIZS/ZnS nanocrystals. Without being limited to a single theory,
it currently is believed that he radiative decay from trap states
or new electron-hole recombination pathways in the Mn-alloyed AISZ
layer could be the dominant mechanisms for the fast decayed
photoluminescence. With respect to A2, it may be relative to the
ratio of the number of Mn pairs or diluted Mn dopants out of the
Mn-doped layer over the number of all Mn dopants (in and out of the
Mn-doped layer). For a low level Mn (0.025 mmol) in the doping
reaction, the Mn spatial distribution in nanocrystals could be more
uniform by diffusion when compared to high Mn levels, and thus the
ratio of the number of Mn dopants out of the doped layer over the
number of Mn dopants in and out of the doped layer (or
alternatively A.sub.2) would be smaller. Notably, all prepared
nanocrystals have their average photoluminescence lifetimes above
one hundred microseconds, and they have good potential to be used
as time-gated or time resolved probes in biosening/imaging.
TABLE-US-00004 TABLE 4 Measured .tau..sub.1 .tau..sub.2
.tau..sub.avg Nanocrystals Wavelength (ms) A.sub.1 (ms) A.sub.2
(ms) 0.025 mmol- 525 nm 21.8 98.9% 332.6 1.1% 66.6 Mn:AIZS/ZnS 575
nm 30.1 98.1% 280.0 1.9% 67.5 625 nm 27.2 99.1% 517.0 0.9% 100.6
700 nm 17.5 99.8% 498.9 0.2% 39.9 0.075 mmol- 550 nm 33.0 97.0%
311.1 3.0% 95.3 Mn:AIZS/ZnS 585 nm 41.2 96.6% 519.1 3.4% 187.2 635
nm 40.1 96.0% 614.2 4.0% 263.5 700 nm 31.0 98.2% 480.8 1.8% 129.1
0.125 mmol- 550 nm 33.4 97.8% 321.1 2.2% 85.1 Mn:AIZS/ZnS 585 nm
41.3 96.7% 509.7 3.3% 178.6 642 nm 42.8 96.8% 496.4 3.2% 169.4 700
nm 32.3 98.2% 264.1 1.8% 62.9
FIGS. 54A-54C include results obtained from analyzing the
fluorescence lifetime of a control (organic dye Coumarin 153 in
ethanol) and two Mn-doped composite embodiments of the present
disclosure, which were prepared by encapsulating Mn-doped AZIS/ZNS
nanocrystals (their average lifetimes at around 0.1 and 1 ms,
respectively) with a zwitterionic polymeric coating as described
herein. This data was obtained using an optical detection system,
which was capable of measuring fluorescence above 520 nm.
In view of the many possible embodiments to which the principles of
the present disclosure may be applied, it should be recognized that
the illustrated embodiments are only preferred examples of the
present disclosure and should not be taken as limiting the scope.
Rather, the scope of the present disclosure is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
* * * * *